Injectable Melphalan Compositions Comprising a Cyclodextrin Derivative and Methods of Making and Using the Same

ABSTRACT

The present invention is directed to pharmaceutical compositions comprising melphalan and a cyclodextrin derivative, and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Application No. 61/182,560, filed May 29, 2009, which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pharmaceutical compositions comprisingmelphalan and a cyclodextrin derivative and methods of making and usingthe same, for example, to treat disorders and diseases that aretherapeutically responsive to melphalan.

2. Background of the Invention

Melphalan is an alkylating agent of the bischloroethylamine type, and isactive against both resting and rapidly dividing tumor cells. See, e.g.,U.S. Pat. Nos. 3,032,584, 3,032,585, and 4,997,651. An injectablemelphalan composition (ALKERAN® for Injection, GlaxoSmithKline) has beenapproved by the U.S. Food and Drug Administration for the palliativetreatment of patients with multiple myeloma for whom oral therapy is notappropriate, and an oral melphalan composition (ALKERAN® Tablets,GlaxoSmithKline) has been approved for the palliative treatment ofmultiple myeloma and for the palliation of non-resectable epithelialcarcinoma of the ovary.

ALKERAN® for Injection (GlaxoSmithKline) is administered intravenouslyafter first diluting a sterile, nonpyrogenic, freeze-dried powdercontaining melphalan hydrochloride (equivalent to 50 mg melphalan) and20 mg povidone with a sterile diluent that contains sodium citrate (0.2g), propylene glycol (6 mL), ethanol (96%, 0.52 mL), and water, for atotal volume of 10 mL. The usual intravenous dose is 16 mg/m², which isadministered as a single infusion over 15 to 20 minutes. Melphalan isintravenously administered in 4 doses at 2-week intervals, then, afteradequate recovery from toxicity, at 4-week intervals.

According to the ALKERAN® for Injection (GlaxoSmithKline) label,following administration of ALKERAN® for Injection, drug plasmaconcentrations of melphalan decline rapidly in a biexponential mannerwith distribution phase and terminal elimination phase half-lives ofapproximately 10 and 75 minutes, respectively. The average total bodyclearance is 7 to 9 mL/min/kg (250 to 325 mL/min/m²). A study hasreported that on repeat dosing of 0.5 mg/kg every 6 weeks, the clearanceof melphalan decreased from 8.1 mL/min/kg after the first course, to 5.5mL/min/kg after the third course, but did not decrease appreciably afterthe third course. Mean (±SD) peak melphalan plasma concentrations inmyeloma patients after administration of 10 or 20 mg/m² doses ofmelphalan were 1.2±0.4 and 2.8±1.9 μg/mL, respectively. Afterintravenous administration of 50 mg of melphalan, the steady-statevolume of distribution of melphalan is 0.5 L/kg. The extent of melphalanbinding to plasma proteins ranges from 60% to 90%. Serum albumin is themajor binding protein, while α₁-acid glycoprotein appears to account forabout 20% of the plasma protein binding. Approximately 30% of the drugis (covalently) irreversibly bound to plasma proteins. Interactions withimmunoglobulins have been found to be negligible.

Melphalan is eliminated from plasma primarily by chemical hydrolysis tomonohydroxymelphalan and dihydroxymelphalan. Aside from these hydrolysisproducts, no other melphalan metabolites have been observed in humans.

Controlled trials comparing intravenous to oral melphalan have showngreater myelosuppression with the intravenously administered melphalan.Furthermore, hypersensitivity reactions, including anaphylaxis, haveoccurred in approximately 2% of patients who have received intravenousmelphalan. Melphalan also undergoes rapid hydrolysis in aqueoussolution. See S. A. Stout et al., Int. J. Pharm. 24:193 (1985).Melphalan in the ALKERAN® For Injection (GlaxoSmithKline) product alsorapidly forms a citrate derivative upon reconstitution and cannot berefrigerated due to precipitation of melphalan from solution.

Melphalan compositions comprising a cyclodextrin derivative as a carrierand/or a diluent are known. See, e.g., N. J. Medlicott, et al., J.Pharm. Sci. 87:1138 (1998), D. Q. Ma et al., Int. J. Pharm. 189:227(1999), D. Q. Ma et al., J. Pharm. Sci. 89:275 (2000), and U.S. Pat.Nos, 4,983,586, 5,024,998, and 6,583,125.

BRIEF SUMMARY OF THE INVENTION

What is needed is a melphalan formulation that can minimize thetoxicology and side-effect profile of intravenous melphalan. What isalso needed is an intravenous melphalan formulation having increasedbioavailability and/or an improved rate of therapeutic onset. Alsoneeded is a melphalan composition suitable for intravenousadministration that is stable under ambient and/or refrigeratedconditions, and can provide fully dissolved melphalan without the needfor organic solubilizers (e.g., ethanol and/or propylene glycol, and thelike). What is also needed is a composition free from components thatrapidly form a derivative with melphalan. What is also needed is amelphalan composition suitable for intravenous administration that hasimproved stability, thereby enabling longer duration infusions, andlengthening the melphalan exposure time that a patient receives from asingle, convenient administration. As described herein, compositionssuitable for oral or parenteral administration that include melphalanand a cyclodextrin derivative have been developed.

The present invention is directed to a pharmaceutical compositioncomprising 25 mg to 125 mg of melphalan as a hydrochloride salt, anoptional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6; whereindilution of the pharmaceutical composition with an aqueous solutionprovides a melphalan solution ready for infusion in which the melphalandegrades by 2% or less at about 25° C. within 5 hours, or 4% or less atabout 25° C. within 10 hours after the dilution; and wherein thecyclodextrin derivative is present in a ratio of 50:1 to 100:1 (w/w)relative to the melphalan.

The present invention is also directed to a pharmaceutical compositioncomprising 150 mg to 250 mg of melphalan as a hydrochloride salt, anoptional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6, whereindilution of the pharmaceutical composition with an aqueous solutionprovides a melphalan solution ready for infusion in which the melphalandegrades by 2% or less at about 25° C. within 5 hours, or 4% or less atabout 25° C. within 10 hours after the dilution, and wherein thecyclodextrin derivative is present in a ratio of 25:1 to 35:1 (w/w)relative to the melphalan.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; and

the pharmaceutical composition comprises about 50 mg of melphalan as ahydrochloride salt and the cyclodextrin derivative is present in aconcentration of 50:1 to 100:1 (w/w) relative to the melphalan; or

the pharmaceutical composition comprises about 50 mg of melphalan as ahydrochloride salt the cyclodextrin derivative is present in a ratio ofabout 55:1 (w/w) relative to the melphalan; or

the pharmaceutical composition comprises about 200 mg of melphalan as ahydrochloride salt and the cyclodextrin derivative is present in a ratioof 25:1 to 35:1 (w/w) relative to the melphalan; or

the pharmaceutical composition comprises about 200 mg of melphalan as ahydrochloride salt and the cyclodextrin derivative is present in a ratioof about 27:1, about 30:1, or about 32:1 (w/w) relative to themelphalan.

The present invention is directed to a method of treating a subjectsuffering from a neoplastic disorder, the method comprising diluting acomposition with an aqueous diluent to provide a dilute pharmaceuticalcomposition comprising 25 mg to 125 mg of melphalan and a cyclodextrinderivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thedilute pharmaceutical composition has a pH of about 4 to about 6;wherein the cyclodextrin derivative is present in a concentration of atleast 50:1 (w/w) relative to the melphalan; wherein the melphalan in thedilute pharmaceutical composition degrades by 2% or less at about 25° C.within 5 hours, or 4% or less at about 25° C. within 10 hours after thediluting; and administering the dilute pharmaceutical composition byinjection to the subject in need thereof.

In some embodiments, the neoplastic disorder is selected from: myeloma,multiple myeloma, acute myelogenous leukemia, melanoma, malignantmelanoma, breast cancer, ovarian cancer, testicular cancer, advancedprostate cancer, a neuroendocrine cancer, metastatic melanoma (e.g.,metastatic ocular melanoma, metastatic cutaneous melanoma, and thelike), a metastatic neuroendocrine tumor, a metastatic adenocarcinomatumor, hepatocellular carcinoma, osteogenic sarcoma, polycythemiaveraplasma, plasma cell neoplasm, amyloidosis, scleromyxedema, andcombinations thereof. In some embodiments, the neoplastic disorder ismultiple myeloma and the administering is systemic and providespalliative treatment of the multiple myeloma.

The present invention is also directed to a method for conditioning asubject in need of a stem cell transplantation, the method comprisingadministering a melphalan dose of 50 mg/m² to 300 mg/m² per day to thesubject in need of the stem cell transplantation, wherein the melphalandose is administered in a pharmaceutical composition comprisingmelphalan and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6; and whereinthe cyclodextrin derivative is present in a ratio of at least 25:1 (w/w)relative to the melphalan.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ is a hydroxy-substituted-C₃ group.

In some embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ areindependently a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ grouphaving a degree of substitution of 4 to 8 per cyclodextrin derivative,and the remaining substituents are —H.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ is substituted with a straight-chain C₄-(alkylene)-SO₃ ⁻ group.

In some embodiments, a pharmaceutical composition or a dilutepharmaceutical composition is substantially free of an alcohol.

In some embodiments, the aqueous diluent is a saline solution.

In some embodiments, a dilute pharmaceutical composition is stored about0.5 hours to about 48 hours prior to the administering. In someembodiments, the melphalan in a pharmaceutical composition of thepresent invention degrades by 2% or less at about 25° C. within 5 hours,or 4% or less at about 25° C. within 10 hours after the diluting.

In some embodiments, a subject suffering from a neoplastic disorder orin need of a stem cell transplantation is a pediatric subject.

In some embodiments, the administering is performed intravenously. Insome embodiments, the administering is performed via a limb perfusion.

In some embodiments, the administering is for a period of two or moredays.

In some embodiments, the administering provides a melphalan C_(max) in asubject that is at least 20% or greater than a melphalan C_(max)provided by a melphalan formulation containing an equivalent dose ofmelphalan and lacking the cyclodextrin derivative. In some embodiments,the administering provides a melphalan AUC_(0-t) in a subject that is atleast 20% or greater than a melphalan AUC_(0-t) provided by a melphalanformulation containing an equivalent dose of melphalan and lacking thecyclodextrin derivative.

In some embodiments, a method of the present invention comprisesdiluting a concentrated melphalan composition with an aqueous diluent toprovide the pharmaceutical composition. In some embodiments, aconcentrated melphalan composition comprises 50 mg to 500 mg ofmelphalan. In some embodiments, a concentrated melphalan compositioncomprises about 200 mg of melphalan.

The present invention is also directed to a pharmaceutical kitcomprising a first container comprising 25 mg to 125 mg of melphalan asa hydrochloride salt and an optional water-soluble polymer, and a secondcontainer comprising an aqueous diluent, an optional buffer, and acyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of at least 50:1 (w/w) relative to the melphalan; andwherein combining the first container and the second container providesa dilute pharmaceutical composition having a pH of about 4 to about 6that degrades by 2% or less at about 25° C. within 5 hours, or 4% orless at about 25° C. within 10 hours after the diluting.

The present invention is also directed to a pharmaceutical kitcomprising a first container comprising 150 mg to 250 mg of melphalan asa hydrochloride salt and an optional water-soluble polymer; and a secondcontainer comprising an aqueous diluent, an optional buffer, and acyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of 25:1 to 35:1 (w/w) relative to the melphalan; andwherein combining the first container and the second container providesa dilute pharmaceutical composition having a pH of about 4 to about 6that degrades by 2% or less at about 25° C. within 5 hours, or 4% orless at about 25° C. within 10 hours after the diluting.

In some embodiments, a first container comprises povidone in an amountof 10 mg to 30 mg, and a second container comprises a pH-adjusting agentin a concentration sufficient to provide a pH of about 4 to about 6 whenthe first container and the second container are combined.

In some embodiments, the cyclodextrin derivative present in the secondcontainer is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; whereinthe first container comprises about 200 mg of melphalan as ahydrochloride salt; and wherein the cyclodextrin derivative is presentin the second container in an amount of about 27:1, about 30:1, or about32:1 (w/w) relative to the melphalan.

Further embodiments, features, and advantages of the present inventions,as well as the composition, structure and operation of the variousembodiments of the present invention, are described in detail below withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, further serve to explainthe principles of the invention and to enable a person skilled in thepertinent art to make and use the invention.

FIG. 1 provides a graphic representation of the solubility of free basemelphalan as a function of pH and the concentration of a cyclodextrinderivative.

FIG. 2 provides a graphic representation of the solubility of free basemelphalan and melphalan hydrochloride at pH 7.5 as a function of theconcentration of a cyclodextrin derivative.

FIGS. 3 and 4 provide flow charts that describe processes for preparinga unit dosage form of the present invention.

FIGS. 5A-5B provide a graphic representation of dose-normalized wholeblood (FIG. 5A) and plasma (FIG. 5B) concentrations of melphalanfollowing intravenous administration to male Sprague-Dawley rats using amelphalan formulation containing a cyclodextrin derivative(SBE_(6.5)-β-CD) and a cyclodextrin-free melphalan formulation (ALKERAN®for Injection, GlaxoSmithKline).

FIG. 6 provides a graphic representation of the mean plasma melphalanconcentration in a human patient after intravenous administration of amelphalan formulation containing a cyclodextrin derivative(SBE_(6.5)-β-CD) and after intravenous administration of acyclodextrin-free melphalan formulation (Melphalan HCl Injectable,Bioniche Pharma USA).

One or more embodiments of the present invention will now be describedwith reference to the accompanying drawings. In the drawings, likereference numbers can indicate identical or functionally similarelements. Additionally, the left-most digit(s) of a reference number canidentify the drawing in which the reference number first appears.

DETAILED DESCRIPTION OF THE INVENTION

This specification discloses one or more embodiments that incorporatethe features of this invention. The disclosed embodiment(s) merelyexemplify the invention. The scope of the invention is not limited tothe disclosed embodiment(s). The invention is defined by the claimsappended hereto.

Throughout the specification, use of the term “about” with respect toany quantity is contemplated to include that quantity. For example,“about 10 mL” is contemplated herein to include “10 mL,” as well asvalues understood in the art to be approximately 10 mL with respect tothe entity described.

The invention includes combinations and sub-combinations of the variousaspects and embodiments disclosed herein. Further, when a particularfeature, structure, or characteristic is described in connection with anembodiment, it is understood that it is within the knowledge of oneskilled in the art to effect such feature, structure, or characteristicin connection with other embodiments whether or not explicitlydescribed. These and other aspects of this invention will be apparentupon reference to the following detailed description, examples, claimsand attached figures.

Melphalan

The compositions, formulations and unit dosage forms of the presentinvention comprise melphalan, which has the following chemicalstructure:

As used herein, the term “melphalan” refers to the L-isomer of the abovecompound, 4-[bis(chloroethyl)amino]phenylalanine, as well as additionsalts, polymorphs, solvates, hydrates, dehydrates, co-crystals,anhydrous, and amorphous forms thereof. Melphalan contains a chiralatom, and thus, as used herein, “melphalan” can refer to thesubstantially pure form of the L-isomer. As used herein, “substantiallypure” refers to melphalan having a purity of 90% or higher, 95% orhigher, 98% or higher, 99% or higher, 99.5% or higher, or 99.9% orhigher.

The D-isomer of the above compound, known as medphalan, is less activeagainst certain animal tumors, and the dose needed to produce effects onchromosomes is larger than that required with melphalan. The racemic(DL-) form is known as merphalan or sarcolysin. In some embodiments, thecompositions of the present invention are substantially free ofmedphalan. In some embodiments, the compositions of the presentinvention comprise melphalan as a hydrochloride salt having a purity of95% or greater, 98% or greater, 99% or greater, 99.9% or greater, or99.99% or greater.

Melphalan is a bifunctional alkylating agent that is active againstselected human neoplastic diseases. The molecular formula for melphalanis C₁₃H₁₈Cl₂N₂O₂, and the molecular weight of the free base form is305.20 g/mol. Melphalan is practically insoluble in water (pH 7) and hasa pK_(a) of about 2.5.

In some embodiments, the pharmaceutical compositions and dosage forms ofthe present invention comprise melphalan as a hydrochloride salt. Asused herein, “melphalan as a hydrochloride salt” refers to thehydrochloric acid addition salt of the above compound. However, amountsand concentrations of melphalan are provided in reference to anequivalent mass of free base melphalan. Thus, 5 mg of “melphalan as ahydrochloride salt” refers to 5 mg of the active agent melphalan,exclusive of the hydrochloride addition salt, which if considered wouldprovide a total mass of about 5.6 mg.

Cyclodextrin Derivatives

The compositions, formulations and/or unit dosage forms of the presentinvention comprise a cyclodextrin derivative. As used herein,“cyclodextrin derivative” refers to a cyclic oligosaccharide comprisingfive or more α-D-glucopyranoside units linked in a circular 1→4configuration, and comprising a substituent group attached to one ormore of the glucopyranoside units at the 2, 3 and/or 6 position(s)through an ether bond (—O—R—, where R refers to the substituent group).

In some embodiments, the cyclodextrin derivative is a compound offormula I:

wherein n is 4, 5 or 6, wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently selected from: —H, a straight-chain or branchedC₁-C₈-(alkylene)-SO₃ ⁻ group, and an optionally substitutedstraight-chain or branched C₁-C₆ group.

In some embodiments, a cyclodextrin for use with the present inventionis selected based upon an average degree of substitution (“ADS”), whichas used herein refers to the average number of substituent groups percyclodextrin molecule. The average degree of substitution forcyclodextrin derivatives is described in detail in WO 2009/018069, whichis incorporated herein by reference in its entirety. As used herein, acyclodextrin derivative composition for use with the present inventionis referred to by the following notation: the substituent(s) areabbreviated (e.g., sulfobutyl ether groups are abbreviated as “SBE”)with a subscript denoting the ADS of the substituent, and cyclodextrinstructure is defined. For example, a sulfobutyl ether-derivatizedβ-cyclodextrin composition having an ADS of 6.5 is referred to as“SBE_(6.5)-β-CD.” As a second example, a β-cyclodextrin compositioncomprising cyclodextrin molecules derivatized with both sulfobutyl etherand hydroxypropyl groups is referred to as “SBE_(4.2)-HP_(2.5)-β-CD,”wherein the ADS of the sulfobutyl ether groups is 4.2 and the ADS of thehydroxypropyl groups is 2.5.

Cyclodextrin derivatives suitable for use with the present inventioninclude cyclodextrin compositions bearing substituent groups (R₁-R₉ andR in formulas I and II, respectively) that are independently selectedfrom: —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group, andan optionally substituted straight-chain or branched C₁-C₆ group.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ of formula I is substituted with a straight-chain C₄-(alkylene)-SO₃ ⁻group. Exemplary C₁-C₈-(alkylene)-SO₃ ⁻ groups suitable for use with thepresent invention include, but are not limited to, sulfoethyl,sulfopropyl, 1-methyl-sulfopropyl, sulfobutyl, 1-methyl-sulfobutyl,2-methyl-sulfobutyl, 1-methyl-sulfobut-3-yl, 2-ethyl-sulfobutyl,3-ethyl-sulfobutyl, sulfopentyl, 1-sulfopent-3-yl, sulfohexyl,sulfoheptyl, sulfooctyl, and the like, and combinations thereof.

In some embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ of formula Iare independently a straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻group having an ADS of 4 to 8, 4 to 7.5, 4 to 7, 4 to 6.5, 4.5 to 8, 4.5to 7.5, 4.5 to 7, 5 to 8, 5 to 7.5, 5 to 7, 5.5 to 8, 5.5 to 7.5, 5.5 to7, 5.5 to 6.5, 6 to 8, 6 7.5, 6 to 7.1, 6.5 to 7, about 6.5, or about 7per cyclodextrin derivative, and the remaining substituents are —H.

In some embodiments, a substituent is an optionally substitutedstraight-chain or branched C₁-C₆ group. As used herein, “optionallysubstituted” refers to one or more optional substituents selected from:halogen (i.e., —F, —Cl, —Br, —I), —NO₂, —C≡N, —OR₂₂, —SR₂₂, —SO₂R₂₂,—C(═O)OR₂₂, —C(═O)R₂₂, —C(═O)N(R₂₂)₂, —SO₂N(R₂₂)₂, —SO₂N(H)C(═O)R₂₂,—SO₂N(H)C(═O)OR₂₂ (wherein R₂₂ is not H), —N(R₂₂)₂, —N(R₂₂)SO₂R₂₂,—N(R₂₂)C(O)_(m)R₂₂ (wherein m=1 or 2), —N(R₂₂)C(O)N(R₂₂)₂,—N(R₂₂)SO₂N(R₂₂)₂, —O—C(═O)R₂₂, —O—C(═OR₂₂, —O—C(═O)N(R₂₂)₂,—C(═O)N(H)SO₂N(R₂₂)₂, —C(═O)N(H)SO₂R₂₂, oxo (or keto, i.e., ═O), thioxo(i.e., ═S), imino (i.e., ═NR₂₂), —NR₂₂—C(—NR₂₂)R₂₂,—NR₂₂—C(═NR₂₂)N(R₂₂)₂, —C(═NR₂₂)N(R₂₂)₂, —O—C(═NR₂₂)N(R₂₂)₂,—O—C(═NR₂₂)R₂₂, —C(═NR₂₂)R₂₂, —C(═NR₂₂)OR₂₂, and ionic forms thereof(e.g., —N⁺(R₂₂)₂X⁻, and the like, wherein X— is a pharmaceuticallyacceptable anion), wherein R₂₂ is independently selected at eachoccurrence from H, and C₁-C₄ alkyl.

Exemplary optionally substituted straight-chain or branched C₁-C₆ groupsinclude, but are not limited to, 2-hydroxypropyl, 3-hydroxypropyl,2,3-dihydroxypropyl, 3-oxobutyl, and 2-ethoxy-ethyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ of formula I is a hydroxy-substituted-C₃ group. In some embodiments,the cyclodextrin derivative comprises β-cyclodextrin that includes ahydroxy-substituted-C₃ group having an ADS of 1 to 8, 2 to 8, 3 to 7, 4to 7.5, 4.3 to 7.5, about 1, about 2, about 2.5, about 3, about 3.5,about 4, about 4.3, about 5, about 5.5, about 6, about 6.5, about 7, orabout 7.5.

Exemplary cyclodextrin compositions, and methods of making the same,that are suitable for use with the present invention also include thosedescribed in U.S. Pat. Nos. 5,134,127, 5,241,059, 5,376,645, 5,874,418,6,046,177, 6,133,248, 6,153,746, 6,204,256, 7,034,013, 7,629,331, and7,635,773, U.S. Pub. No. 2009/0012042, and PCT Pub. No. WO 2005/117911,the contents of each of which is incorporated herein by reference in theentirety.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x). In some embodiments,x=6.0-7.1. In some embodiments, the cyclodextrin derivative of formulaII has an average molecular weight of about 2163 g/mol.

In some embodiments, the cyclodextrin derivative is a sulfobutylether-β-cyclodextrin having an ADS of about 7 (e.g., CAPTISOL®, CyDexPharmaceuticals, Inc., Lenexa, Kans.). CAPTISOL® cyclodextrin is apolyanionic β-cyclodextrin derivative with a sodium sulfonate saltseparated from the lipophilic cyclodextrin cavity by a butyl etherspacer group, or sulfobutylether (SBE). CAPTISOL® cyclodextrin has beenshown to be safe when administered parenterally, orally, or viainhalation and does not exhibit the nephrotoxicity associated withβ-cyclodextrin. Relative to β-cyclodextrin, CAPTISOL® sulfoalkyl ethercyclodextrin provides comparable or higher complexation characteristicsand superior water solubility in excess of 90 g per 100 mL, a 50-foldimprovement. Melphalan has a low binding affinity with CAPTISOL®(K_(a)=3×10² M⁻¹).

In some embodiments, the cyclodextrin derivative includes a substituentthat bears an ionic group that can optionally form a salt with apharmaceutically acceptable anion or cation. Pharmaceutically acceptablecations suitable for forming salts with negatively charged cyclodextrinderivatives of the present invention include, but are not limited to,H⁺, Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, ammonium and amine cations such as cationsof (C₁-C₆)-alkylamines, (C₄-C₈)-cycloalkylamines (e.g., piperidine,pyrazine, and the like), (C₁-C₆)-alkanolamines, and(C₄-C₈)-cycloalkanolamines, and the like, and combinations thereof. Insome embodiments, a pharmaceutically acceptable cation is Na⁺.Pharmaceutically acceptable anions suitable for forming salts withpositively charged cyclodextrin derivatives of the present inventioninclude, but are not limited to, halides (e.g., Cl⁻ and the like),anions of (C₁-C₆)-alkyl acids (e.g., acetate, oxalate, fumarate,succinate, and the like, and combinations thereof.

Pharmaceutical Compositions and Unit Dosage Forms

The present invention is directed to pharmaceutical compositions andunit dosage forms comprising melphalan and a cyclodextrin derivative.The pharmaceutical compositions of the present invention are suitablefor parenteral administration to a subject. Parenteral administration ofthe pharmaceutical compositions can include, but is not limited to, aninjection. Because parenteral administration bypasses a subject'snatural defenses against contaminants, the pharmaceutical compositionsare sterile or capable of being sterilized prior to administration.

Exemplary pharmaceutical compositions include, but are not limited to,solutions, suspensions or emulsions ready for administration, solutions,suspensions or emulsions ready to be dissolved in and/or diluted with apharmaceutically acceptable vehicle, and dry products ready to bedissolved in and/or diluted with a pharmaceutically acceptable vehicle.

Generally, the pharmaceutical compositions of the present inventioncomprise melphalan in a concentration suitable for treating a conditionthat is amenable to treatment with melphalan. Thus, the pharmaceuticalcompositions of the present invention can be used to prepare a unitdosage form comprising a therapeutically effective amount of melphalanfor administering to a subject in need thereof. In some embodiments, thepresent invention is directed to a unit dosage form that comprisesmelphalan in a concentration that is suitable for administration withoutdilution. Alternatively, a unit dosage form of the present invention canbe diluted prior to administration to a subject in need thereof.

The present invention is also directed to a pharmaceutical compositioncomprising 25 mg to 125 mg, 25 mg to 100 mg, 25 mg to 75 mg, 25 mg to 50mg, 50 mg to 125 mg, 50 mg to 100 mg, 75 to 125 mg, 100 to 125 mg, about25 mg, about 50 mg, about 75 mg, about 100 mg, or about 125 mg ofmelphalan as a hydrochloride salt, an optional buffer, and acyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6, about 4 toabout 5, about 4.5 to about 6, about 5 to about 6, about 5.5 to about 6,about 4, about 4.5, about 5, about 5.5, or about 6; wherein dilution ofthe pharmaceutical composition with an aqueous solution provides asolution in which the melphalan degrades by 2% or less at about 25° C.within 5 hours, or by 4% or less at about 25° C. within 10 hours afterthe dilution; and wherein the cyclodextrin derivative is present in aratio of 50:1 to 100:1, 55:1 to 60:1, about 50:1, about 55:1, or about60:1 (w/w) relative to the melphalan.

The present invention is also directed to a pharmaceutical compositioncomprising 150 mg to 300 mg, 150 mg to 250 mg, 150 mg to 225 mg, 175 mgto 250 mg, 200 mg to 250 mg, about 150 mg, about 175 mg, about 200 mg,about 225 mg, or about 250 mg of melphalan as a hydrochloride salt, anoptional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independent;y —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6, whereindilution of the pharmaceutical composition with an aqueous solutionprovides a melphalan solution in which the melphalan degrades by 2% orless at about 25° C. within 5 hours, or 4% or less at about 25° C.within 10 hours after the dilution; and wherein the cyclodextrinderivative is present in a ratio of 25:1 to 35:1, about 27:1, about30:1, or about 32:1 (w/w) relative to the melphalan.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1.

In some embodiments, the cyclodextrin derivative is a compound offormula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; and

the pharmaceutical composition comprises about 50 mg of melphalan as ahydrochloride salt and the cyclodextrin derivative is present in aconcentration of 50:1 to 100:1, 55:1 to 60:1, about 50:1, about 55:1, orabout 60:1 (w/w) relative to the melphalan; or

the pharmaceutical composition comprises about 200 mg of melphalan as ahydrochloride salt and the cyclodextrin derivative is present in a ratioof 25:1 to 35:1, about 27:1, about 30:1, or about 32:1 (w/w) relative tothe melphalan.

Sterile solutions, suspensions, emulsions and the like can be preparedby incorporating melphalan into an appropriate solvent or carrier withthe other optional ingredients enumerated herein, followed bysterilization. Sterile powders can be prepared by spray drying, asepticspray drying, vacuum drying, or freeze drying a sterile solution,suspension, or emulsion to provide a dried solid (e.g., a powder)comprising melphalan along with any additional excipients.

In some embodiments, the present invention is directed to a solidpharmaceutical composition consisting of about 50 mg of melphalan as ahydrochloride salt, an amount sufficient of an acid, a base, or acombination thereof to provide a pH of about 4 to about 6 upon dilutionwith a saline solution to a volume of about 10 mL, and a cyclodextrinderivative of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1, whereindilution of the solid pharmaceutical composition with an aqueoussolution provides a melphalan solution in which the melphalan degradesby 2% or less at about 25° C. within 5 hours, or by 4% or less at about25° C. within 10 hours after the diluting, and wherein the cyclodextrinderivative is present in a ratio of about 55:1 (w/w) relative to themelphalan.

In some embodiments, the present invention is directed to a solidpharmaceutical composition consisting of about 200 mg of melphalan as ahydrochloride salt, an amount sufficient of an acid, a base, or acombination thereof to provide a pH of about 4 to about 6 upon dilutionwith a saline solution to a volume of about 20 mL, and a cyclodextrinderivative of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1, whereindilution of the solid pharmaceutical composition with an aqueoussolution provides a melphalan solution in which the melphalan degradesby 2% or less at about 25° C. within 5 hours, or by 4% or less at about25° C. within 10 hours after the diluting, and wherein the cyclodextrinderivative is present in a ratio of about 27:1, about 30:1, or about32:1 (w/w) relative to the melphalan.

In some embodiments, a pharmaceutical composition or unit dosage form ofthe present invention comprises a solid (e.g., a powder) or a liquidsolution that is diluted with a liquid carrier or diluent prior toadministration to a subject. Thus, the pharmaceutical compositions andunit dosage forms of the present invention include sterile aqueoussolutions, suspensions and dispersions, as well as sterile solids (e.g.,powders) comprising melphalan that can be extemporaneously diluted orsolubilized to provide a sterile solution, suspension or dispersion.

In some embodiments, the compositions, formulations and/or unit dosageforms of the present invention comprise a pharmaceutically acceptableexcipient. As used herein, “pharmaceutically acceptable” refers to thoseexcipients, compounds, materials, and/or compositions which are, withinthe scope of sound medical judgment, suitable for contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other possible complicationscommensurate with a reasonable benefit/risk ratio.

In some embodiments, the pharmaceutical compositions and unit dosageforms of the present invention are substantially homogeneous. As usedherein, “homogeneous” refers to mixtures, solutions, suspensions,compositions, dosage forms, and/or formulations of the present inventionthat have a uniform distribution of ingredients throughout. Homogeneityis synonymous with uniformity and can refer to intra-sample uniformity,batch-to-batch uniformity, run-to-run uniformity, and/or dosageform-to-dosage form uniformity. For example, intra-sample uniformity canbe determined by analyzing a first portion of a sample, mixture, orcomposition and comparing this with a second portion of the same sample,mixture, or composition. Typical deviations of a composition (e.g.,variation in the percentage by weight of excipients and the like) of asubstantially homogeneous composition are about 5% or less, about 3% orless, about 2% or less, about 1% or less, or within experimental error.

In some embodiments, a pharmaceutical composition or unit dosage form ofthe present invention comprises a pharmaceutically acceptable excipient.As used herein, the term “excipient” refers to any inert substance thatcan be combined with melphalan and the sulfoalkyl ether cyclodextrin forpreparing the pharmaceutical compositions.

Pharmaceutically acceptable excipients suitable for use with the presentinvention include, but are not limited to, a carrier, a water-solublepolymer, a preservative, an antioxidant, a pH-adjusting agent (e.g., anacidifying agent, an alkalinizing agent, and/or a buffer), a bulkingagent, a complexation enhancing agent, a cryoprotectant, a densitymodifier, an electrolyte, a flavor, a fragrance, a lyophilizing aid(e.g., a bulking agent and/or stabilizing agent), a plasticizer, asolubility-enhancing agent, a stabilizing agent, a sweetener, a surfacetension modifier, a volatility modifier, a viscosity modifier, andcombinations thereof. In addition, one of skill in the art willrecognize that pharmaceutically acceptable excipients can be used in thepresent invention including those listed in The Handbook ofPharmaceutical Excipients, 5th Ed., The Pharmaceutical Press andAmerican Pharmacists Association, London, UK and Washington, D.C.(2006), which is incorporated herein by reference in its entirety.

In some embodiments, a pharmaceutical composition or unit dosage form ofthe present invention comprises a pharmaceutically acceptable carrier.As used herein, a “carrier” refers to a vehicle suitable fortransferring and/or diluting a pharmaceutical composition or unit dosageform of the present invention. Pharmaceutically acceptable carrierssuitable for use with the present inventions include, but are notlimited to, liquids, solids, colloids, gels, and combinations thereof.Liquid carriers suitable for use with the present invention includesolvents, liquid dispersion mediums, and the like, such as, but notlimited to, water, ethanol, a polyol (e.g., glycerol, propylene glycol,liquid polyethylene glycols, and the like), a vegetable oil, a nontoxicglyceryl ester, and combinations thereof. In some embodiments, a liquidcarrier is selected from: a dextrose solution, a saline solution,plasma, and lactated Ringer's solution.

In some embodiments, a pharmaceutical composition or unit dosage form ofthe present invention comprises a water-soluble polymer such as, but notlimited to, homopolymers of N-polyvinylpyrrolidone (e.g., “povidone”),low molecular weight hydroxypropyl cellulose, low molecular weightmethyl cellulose, low molecular weight hydroxypropyl methyl cellulose,and the like, and combinations thereof.

In some embodiments, after dilution a cyclodextrin derivative is presentin the diluted pharmaceutical composition in a concentration of about 75mM, about 100 mM, or about 125 mM.

In some embodiments, after dilution melphalan is present in the dilutedpharmaceutical composition in a concentration of 0.1 mg/mL to 50 mg/mL,0.15 mg/mL to 40 mg/mL, 0.2 mg/mL to 30 mg/mL, 0.3 mg/mL to 25 mg/mL,0.4 mg/mL to 20 mg/mL, 0.45 mg/mL to 15 mg/mL, 0.5 mg/mL to 10 mg/mL,about 0.45 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about2.5 mg/mL, or about 5 mg/mL.

In some embodiments, a pharmaceutical composition and/or a diluent foruse with a composition of the present invention is free of asolubilizing agent such as, but not limited to, water, an alcohol (e.g.,ethanol and the like), a polyol (e.g., glycerol, propylene glycol,liquid polyethylene glycols, and the like), a vegetable oil, a nontoxicglyceryl ester, and combinations thereof. Thus, in some embodiments thediluent consists essentially of water and optional tonicity-adjustingagents (e.g., 0.9% saline solution for injection, and the like).

In some embodiments, the pH of a pharmaceutical composition or unitdosage form is controlled. In some embodiments, a pharmaceuticalcomposition or unit dosage form of the present invention comprises apharmaceutically acceptable buffer and/or pH adjusting agent (e.g., anacidifying agent and/or alkalinizing agent). In some embodiments, apharmaceutical composition or unit dosage form of the present inventionhas a pH of about 4 to about 6, about 4 to about 5, about 5 to about 6,about 4, about 5, about 5.5, or about 6 after dilution with an aqueousdiluent.

In some embodiments, a pharmaceutical composition or unit dosage formthat is to be diluted prior to administration to a subject has a pH ofabout 2 to about 6, about 3 to about 6, about 4 to about 6, or about 5to about 6. In some embodiments, after dilution (e.g., with an liquidcarrier) a unit dosage form of the present invention has a pH of about 4to about 6, about 4 to about 5, about 5 to about 6, about 4, about 4.5,about 5, about 5.5, or about 6 at the time of administration to asubject in need thereof.

In some embodiments, a pharmaceutical composition or unit dosage form ofthe present invention comprises a buffer. In some embodiments, apharmaceutical composition or unit dosage form of the present inventioncomprises a buffer suitable to provide a dilute composition having a pHof about 4 to about 6, about 4 to about 5, about 5 to about 6, about 4,about 4.5, about 5 about 5.5, or about 6. In some embodiments, a bufferis present in a concentration of about 0.01 M to about 10 M, about 0.01M to about 5 M, or about 0.01 M to about 1 M.

In some embodiments, a pharmaceutical composition or unit dosage form ofthe present invention comprises a pH-adjusting agent such as, but notlimited to, an acidifying agent (e.g., citric acid, HCl, and the like),an alkalinizing agent (e.g., NaOH and the like), a salt form of an acid(e.g., sodium citrate and the like), and combinations thereof. In someembodiments, a pharmaceutical composition or unit dosage form of thepresent invention comprises a pH-adjusting agent in an amount sufficientto provide a dilute composition having a pH of about 4 to about 6, about4 to about 5, about 5 to about 6, about 4, about 4.5, about 5 about 5.5,or about 6. In some embodiments, In some embodiments, a pharmaceuticalcomposition or unit dosage form of the present invention comprisessodium citrate in an amount of 50 mg to 500 mg, 75 mg to 400 mg, 100 mgto 300 mg, 150 mg to 250 mg, or about 200 mg.

In some embodiments, a pharmaceutical composition or unit dosage form ofthe present invention comprises a second therapeutic agent. Suitablesecond therapeutic agents include, but are not limited to, a platinumcompound, an antimetabolite, a nitrosourea, a corticosteroid, acalcineurin inhibitor, a monoclonal antibody, a polyclonal antibody, acytotoxic antibiotic, an interferon, an opioid, an antihistamine, avolume expander, a pressor agent, and combinations thereof. Additionalsecond therapeutic agents include, but are not limited to, doxorubicin,bortezomib, rituximab, thalidomide, lenalidomide, gemcitabine, thiotepa,fludarabine, carmustine, etoposide, cytarabine, granulocytecolony-stimulating factor, ADH-1, topotecan, palifermin, prednisone,arsenic trioxide, ascorbic acid, busulfan, buthionine sulfoximine, andcombinations thereof.

As used herein, a “unit dosage form” refers to a composition containinga specific amount of melphalan, the whole of which is intended to beadministered to a subject in a single dose. A unit dosage form can bedistinguished from a supply of a multi-dose amount of a pharmaceuticalcomposition, e.g., a bottle of medicine, from which a unit dose ismeasured out.

In some embodiments, a unit dosage form of the present inventioncomprises a therapeutically effective amount of melphalan. As usedherein, a “therapeutically effective amount” refers to an amount ofmelphalan that elicits a biological or medicinal response in a tissue,system, animal or human that is being sought by a researcher,veterinarian, medical doctor or other clinician, which includesalleviation of the symptoms of a disease or disorder being treated.

A unit dosage form typically comprises the pharmaceutical composition ofthe present invention and optionally, one or more pharmaceuticallyacceptable excipients, wherein the amount of melphalan present in theunit dosage form is sufficient for a single administration to a subjectin need thereof. Unit dosage forms of the present invention include, butare not limited to, liquid solutions, liquid suspensions, liquiddispersions, emulsions, gels, powders, tablets, capsules, caplets, andthe like. Treatment of a disease or condition amenable to treatment withmelphalan can comprises periodic administration of a unit dosage form ofthe present invention, for example, once every two weeks, once everyfour weeks, or some other interval.

In some embodiments, a unit dosage form of the present inventioncomprises 25 mg to 125 mg, or 150 mg to 250 mg of melphalan as ahydrochloride salt. In some embodiments, a unit dosage form of thepresent invention comprises 50 mg or 200 mg melphalan as a hydrochloridesalt.

In some embodiments, a unit dosage form of the present invention is asolid. In some embodiments, a solid unit dosage form of the presentinvention is a lyophilized solid or an aseptic spray-dried solid. Insome embodiments, a dosage form of the present invention is suitable fordilution and/or reconstitution with a predetermined amount of a liquidcarrier. For example, a unit dosage form (e.g., a liquid or a solid) ofthe present invention can be diluted with 5 mL to 500 mL, 10 mL to 100mL, or 10 mL to 50 mL of a liquid carrier.

The pharmaceutical compositions and unit dosage forms of the presentinvention are stable. As used herein, stability can refer to either theshelf-life of an undiluted solid or liquid dosage form or the resistanceto degradation of a diluted liquid dosage form. In particular, currentlyavailable melphalan compositions suitable for intravenous administrationmust be used as soon as possible after dilution due to the rapiddegradation of melphalan in aqueous solution. However, the dosage formsof the present invention are stable for a considerable time period afterdilution, for example, at least 90 minutes up to at least 48 hours ormore. Thus, in those embodiments in which a solid or liquid unit dosageform is diluted, the diluting can be performed immediately prior toadministering, or sometime before the administering without anysignificant loss of therapeutic efficacy. This enables a liquidpharmaceutical composition or liquid unit dosage form of the presentinvention to be diluted 90 minutes to 48 hours in advance of use (i.e.,in advance of parenteral administration to a subject in need thereof).

In some embodiments, the melphalan in a pharmaceutical composition ofthe present invention degrades by 2% or less at about 25° C. within 5hours, or by 4% or less at about 25° C. within 10 hours after dilutionwith an aqueous diluent to provide a diluted composition comprising acyclodextrin derivative in a concentration of about 75 mM or about 125mM.

The primary degradation product of melphalan in aqueous solution ismelphalan monohydroxide (also known as monohydroxymelphalan), whichproceeds via a hydrolysis reaction. See, e.g., S. A. Stout et al., Int.J. Pharm. 24:193 (1985). In some embodiments, dilution of apharmaceutical composition of the present invention provides a melphalanmonohydroxide concentration (based on a 100% initial concentration ofmelphalan) of 2% or less within 5 hours of the diluting, when thediluted composition is maintained at room temperature (about 25° C.). Insome embodiments, dilution of a pharmaceutical composition of thepresent invention provides a melphalan monohydroxide concentration(based on a 100% initial concentration of melphalan) of 4% or lesswithin 10 hours of the diluting, when the diluted composition ismaintained at room temperature (about 25° C.). In some embodiments,dilution of a pharmaceutical composition of the present inventionprovides a melphalan monohydroxide concentration (based on a 100%initial concentration of melphalan) of 2% or less within 24 hours of thediluting, or 4% or less within 48 hours of the diluting when the dilutedcomposition is maintained at a temperature of about 10° C. or less.

Furthermore, the pharmaceutical compositions of the present inventioncan be stored prior to dilution for an extended period of time withoutany significant loss of melphalan. For example, a solid pharmaceuticalcomposition comprising melphalan and a cyclodextrin derivative contains2% or less, by weight, of a melphalan degradant after storage at 25° C.for a period of at least 2 years, or 5% or less, by weight, of amelphalan degradant after storage at 25° C. for a period of at least 3years.

In some embodiments, a dry powder pharmaceutical composition of thepresent invention forms 2% or less of melphalan monohydroxide (based ona 100% initial concentration of melphalan) after storage for 2 years atroom temperature.

Pharmaceutical Kits

The present invention is also directed to a pharmaceutical kitcomprising a first container comprising 25 mg to 125 mg of melphalan asa hydrochloride salt and an optional water-soluble polymer, and a secondcontainer comprising an aqueous diluent, an optional buffer, and acyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of at least 50:1 (w/w) relative to the melphalan; andwherein combining the first container and the second container providesa dilute pharmaceutical composition having a pH of about 4 to about 6that degrades by 2% or less at about 25° C. within 5 hours after thediluting.

The present invention is also directed to a pharmaceutical kitcomprising a first container comprising 150 mg to 250 mg of melphalan asa hydrochloride salt and an optional water-soluble polymer; and a secondcontainer comprising an aqueous diluent, an optional buffer, and acyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of 25:1 to 35:1 (w/w) relative to the melphalan; andwherein combining the first container and the second container providesa dilute pharmaceutical composition having a pH of about 4 to about 6that degrades by 2% or less at about 25° C. within 5 hours after thediluting.

Alternatively, a first container comprises melphalan (in an amountdescribed above) and a cyclodextrin derivative (as described above), anoptional water-soluble polymer (e.g., povidone and the like), and anoptional pH-adjusting agent; and a second container comprises a diluent(e.g., water, saline, and the like), an optional tonicity adjustingagent, and an optional pH-adjusting agent.

Materials suitable for use as the containers with the kits of thepresent invention include, but are not limited to, a glass (e.g.,borosilicate glass, amber glass, and the like), a plastic (e.g.,polypropylene, high-density polyethylene, poly(ethylene terephthalate,polystyrene, polycarbonate, and the like, and combinations thereof), ametal (e.g., a foil), and the like, and combinations thereof (e.g., aplastic-coated glass and/or metal).

Containers suitable for use with the pharmaceutical kits of the presentinvention include, but are not limited to, vials, bottles, sachets, andthe like. The containers can be opened and/or the contents can beremoved therefrom, by, for example, tearing, cutting, removing ascrew-top, removing a stopper, piercing, squeezing, and the like, andcombinations thereof.

In some embodiments, a first container comprises povidone in an amountof 10 mg to 30 mg, 15 mg to 25 mg, or about 20 mg. In some embodiments,a second container comprises a pH-adjusting agent (e.g., an acidifyingagent, an alkalinizing agent, and/or a buffer) in a concentrationsufficient to provide a pH of about 4 to about 6 when the firstcontainer and the second container are combined. In some embodiments, asecond container comprises sodium citrate in an amount of 50 mg to 500mg, 75 mg to 400 mg, 100 mg to 300 mg, 150 mg to 250 mg, or about 200mg.

In some embodiments, the cyclodextrin derivative present in the secondcontainer is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; whereinthe first container comprises about 200 mg of melphalan as ahydrochloride salt; and wherein the cyclodextrin derivative is presentin the second container in an amount of about 27:1, about 30:1, or about32:1 (w/w) relative to the melphalan.

Methods of Administering and Treating

In some embodiments, the present invention is directed to methods ofdelivering melphalan to a subject in need thereof, the method comprisingadministering a pharmaceutical composition or unit dosage form of thepresent invention to the subject in need thereof. The methods of thepresent invention include parenteral administration of thepharmaceutical compositions or unit dosage forms of the presentinvention.

In some embodiments, the pharmaceutical compositions or unit dosageforms (or diluted forms thereof) are intravenously administered.Intravenous administration includes, but is not limited to, a bolusinjection, an intravenous infusion, a limb perfusion, a normothermicisolated limb infusion, a percutaneous hepatic perfusion, and the like,and combinations thereof. Administering the compositions of the presentinvention can also be performed by injection and/or drip line using acannula, a central line, a peripherally inserted central catheter line,and the like.

In some embodiments, a pharmaceutical composition of the presentinvention is administered as an infusion for a duration of 15 minutes to6 hours, 30 minutes to 4 hours, 45 minutes to 3 hours, 1 hour to 2hours, about 15 minutes, about 30 minutes, about 45 minutes, about 1hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours,about 4 hours, about 5 hours, or about 6 hours.

In some embodiments, the present invention is directed to parenterallyadministering a pharmaceutical composition or unit dosage form of thepresent invention to a subject for which an oral composition ofmelphalan is, for one or more reasons, not appropriate. For example,oral compositions of melphalan may not be appropriate because a subjectmay be too young, unable to swallow, undergoing surgery, incapacitated,or have a disorder that blocks absorption of melphalan administered viathe oral route. Further, parenteral administration of the pharmaceuticalcompositions of the present invention are useful for treating conditionsin subject in which a rapid increase in the in vivo concentration ofmelphalan is required.

In some embodiments, the present invention is directed to a method oftreating and or preventing diseases in a human subject by administeringthe pharmaceutical compositions and or unit dosage forms of the presentinvention to the human subject. In some embodiments, the presentinvention is directed to methods of treating a subject suffering from adisease or disorder amenable to treatment with melphalan, the methodcomprising administering a pharmaceutical composition or unit dosageform to the subject. As used herein the terms “treat,” “treating,” and“treatment” refer to administering a composition of the presentinvention prior to the onset of clinical symptoms of a diseasestate/condition so as to prevent the development of any symptom, as wellas administering the composition after the onset of one or more clinicalsymptoms of a disease state/condition so as to reduce or eliminate anysuch symptom, aspect or characteristic of the disease state/condition.Such treating need not be absolute to be useful. Additionally, the terms“treat” and “treatment” refer to both therapeutic treatment andprophylactic, maintenance, or preventative measures, wherein the objectis to prevent or slow down (lessen) an undesired physiologicalcondition, disorder or disease, or obtain beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of a symptom or asign; diminishment of extent of a condition, disorder or disease;stabilization (i.e., not worsening) of the state of condition, disorderor disease; delay in onset or slowing of condition, disorder or diseaseprogression; amelioration of a condition, disorder or disease state,remission (whether partial or total), whether detectable orundetectable; or enhancement or improvement of condition, disorder ordisease. Treatment includes eliciting a clinically significant response,without excessive levels of side effects. Treatment also includesprolonging survival as compared to expected survival if not receivingtreatment.

As used herein, the term “subject” refers to warm blooded animals suchas mammals, including humans and non-humans, such as, but not limitedto, domestic and farm animals, zoo animals, sports animals, and pets(e.g., cats, dogs, mice, guinea pigs, horses, bovine cows, and sheep).In some embodiments, a subject is a human subject. Human subjectssuitable for administering the pharmaceutical compositions and unitdosage forms of the present invention include, but are not limited to,pediatric, adult, and geriatric subjects. In some embodiments of theinvention, the subject is a pediatric subject. For example, according tothe U.S. Food and Drug Administration, a “pediatric” subject is up to 21years of age, and includes neonates (birth to about 1 month of age),infants (about 1 month to about 2 years of age), children (about 2 toabout 12 years of age) and adolescents (about 12 to about 21 years ofage). See Guidance for Industry and FDA Staff, Premarket Assessment ofPediatric Medical Devices, U.S. Dept. of Health and Human Services, Foodand Drug Administration, Center for Devices and Radiological Health, andCenter for Biologics Evaluation and Research (May 14, 2004). In someembodiments of the invention, the subject is an adult. As used herein,an “adult” subject is 18 years of age or older. In some embodiments, asubject is an adult that is about 50 years or older. In some embodimentsof the invention, the subject is geriatric. Geriatric subjects are atleast about 65 years of age. In some embodiments, a subject is about 70years of age or older.

In some embodiments, the subject is a pediatric subject suffering from adisorder such as, but not limited to, an inborn defect, animmunodeficiency, a combined immunodeficiency, a severe combinedimmunodeficiency, a congenital neutropenia with defective stem cells,aplastic anemia, and combinations thereof.

In some embodiments, a subject is a geriatric subject scheduled toundergo a non-myeloablative procedure.

In some embodiments, the present invention comprises a method fortreating a subject who has or is at risk for developing a conditionamenable to treatment with melphalan, the method comprisingadministering an effective amount (i.e., a therapeutically effectiveamount) of a composition of the invention to the subject. Conditionsamenable to treatment with melphalan include, but are not limited to,neoplastic disorders.

In some embodiments, a therapeutically effective amount foradministering to a subject who has or is at risk for developing acondition amenable to treatment with melphalan is 25 mg to 125 mg, 40 mgto 110 mg, 40 mg to 75 mg, 40 mg to 60 mg, about 40 mg, about 50 mg,about 60 mg, about 75 mg, or about 100 mg of melphalan as ahydrochloride salt. The methods of the present invention also includetitrating upward or downward from an initial melphalan dose in order toprovide a therapeutically effective melphalan dosage. A therapeuticallyeffective dose can be administered once, twice, thrice, four times, fivetimes, six times, seven times, eight times, ten times, twelve times, ormore as needed.

In some embodiments, the present invention is directed to a method oftreating a disease, disorder or condition that is therapeuticallyresponsive to a stem cell transplantation, the method comprisingadministering to a subject in need thereof a pharmaceutical compositionor unit dosage form of the present invention followed by the subjectundergoing a stem cell transplantation.

In some embodiments, a method of the present invention comprisesadministering a pharmaceutical composition or unit dosage form (ordiluted form thereof) to a subject that suffers from a disorder selectedfrom: myeloma, multiple myeloma, acute myelogenous leukemia, malignantmelanoma, metastatic melanoma (e.g., metastatic ocular melanoma,metastatic cutaneous melanoma, and the like), breast cancer, ovariancancer, testicular cancer, advanced prostate cancer, a myelodysplasticsyndrome, a neuroendocrine cancer (e.g., a metastatic neuroendocrinetumor, and the like), a metastatic adenocarcinoma tumor, ahepatocellular carcinoma, osteogenic sarcoma, polycythemia veraplasma,plasma cell neoplasm, amyloidosis, scleromyxedema, and combinationsthereof.

In some embodiments, a method of the present invention comprisesadministering a pharmaceutical composition or unit dosage form (ordiluted form thereof) to a subject for whom a stem cell transplantationhas been indicated (e.g., a hematopoietic stem cell transplantation). Insome embodiments, a subject for whom a stem cell transplantation hasbeen indicated suffers from a disease or disorder selected from: aleukemia, a cancer, a non-malignant disease, and combinations thereof.In some embodiments, a subject for whom a stem cell transplantation hasbeen indicated suffers from a disease or disorder selected from:myeloma, multiple myeloma, a lymphoma, non-Hodgkin lymphoma (“NHL”),leukemia, acute myeloid leukemia (“AML”), Hodgkin's disease, acutelymphoblastic leukemia (“ALL”), a myelodysplastic syndrome (“MDS”), amyeloproliferative disorder (“MPD”), chronic myelogenous leukemia(“CML”), neuroblastoma, aplastic anemia, chronic granulocytic leukemia,a neuroblastoma, sickle-cell disease, osteogenic sarcoma, Ewing'ssarcoma, a desmoplastic small round cell tumor, plasma cell neoplasm,amyloidosis, scleromyxedema, and combinations thereof. In someembodiments, a subject for whom a stem cell transplantation has beenindicated is a subject who would not benefit from prolonged treatmentwith, or is already resistant to, chemotherapy.

Thus, the pharmaceutical compositions and unit dosage forms of thepresent invention are useful for treatment of a condition amenable totreatment with melphalan, as well as use for conditioning a subject inneed thereof for receiving a stem cell transplantation.

The amount of the pharmaceutical composition that is administered istherapeutically effective for the treatment that is desired. Forexample, a therapeutically effective amount for the treatment ofmultiple myeloma refers to an amount which, when administered,diminishes one or more symptoms associated with this disorder.

In some embodiments, the present invention is directed to a method ofconditioning a subject in order to conduct a stem cell transplantation,the method comprising administering an effective amount of apharmaceutical composition or unit dosage of the invention (e.g.,intravenously) to the subject. Thus, the pharmaceutical compositions andunit dosage forms of the present invention are useful for treating asubject who suffers from a condition amenable to treatment by a stemcell transplant. As used herein, “stem cell transplantation” includesautologous and/or allogenic transplantation procedures.

The pharmaceutical compositions of the present invention are suitablefor administering melphalan in a “high-intensity” or myeloblativeconditioning regimen in preparation for a stem cell transplantation, orin a “reduced intensity” conditioning regimen in preparation for a stemcell transplantation. As used herein, “reduced intensity” conditioningrefers to dosages in which a melphalan dose of less than 150 mg/m² isadministered to a subject at any one dose. In some embodiments, thepharmaceutical composition of the present invention is administered to asubject that is 50 years of age or older who suffers from a conditionamenable to treatment by a stem cell transplant.

In some embodiments, the melphalan is administered to a subject in needof a stem cell transplantation at a dose of 50 mg/m² to 300 mg/m2, 50 mg/m² to 250 mg/m², 50 mg/m² to 225 mg/m², 50 mg/m² to 200 mg/m², 50mg/m² to 175 mg/m², 50 mg/m² to 150 mg/m², 100 mg/m² to 300 mg/m², 100mg/m² to 250 mg/m², 100 mg/m² to 225 mg/m², 100 mg/m² to 200 mg/m², 100mg/m² to 175 mg/m², 100 mg/m² to 150 mg/m², 125 mg/m² to 300 mg/m², 125mg/m² to 250 mg/m², 125 mg/m² to 225 mg/m², 125 mg/m² to 200 mg/m², 150mg/m² to 300 mg/m², 150 mg/m² to 250 mg/m², 200 mg/m² to 300 mg/m², 200mg/m² to 250 mg/m², about 50 mg/m², about 100 mg/m², about 125 mg/m²,about 150 mg/m², about 175 mg/m², about 200 mg/m², about 250 mg/m², orabout 300 mg/m².

In some embodiments, the administering comprises a dosage administeredat four week intervals. In some embodiments, the dosage is administeredtwice, thrice, four times, five times, six times, eight times, or tentimes. For example, in some embodiments a dose of about 100 mg/m² isadministered three times with a four-week interval between the doses. Insome embodiments, a dose of about 200 mg/m² is administered twice with afour-week interval between the doses. The final dose can be followed bya stem cell transplantation.

The pharmaceutical compositions and unit dosage forms of the presentinvention can be administered alone or in conjunction with othermedications or pharmaceutical compositions. In some embodiments, amethod of the present invention comprises to a subject a secondtherapeutic agent selected from: an alkylating agent other thanmelphalan, a platinum compound, an antimetabolite, a nitrosourea, acorticosteroid, a calcineurin inhibitor, a monoclonal antibody, apolyclonal antibody, a cytotoxic antibiotic, an interferon, an opioid,an antihistamine, a volume expander, a pressor agent, and combinationsthereof. Additional second therapeutic agents include, but are notlimited to, cisplatin, carboplatin, doxorubicin, bortezomib, rituximab,thalidomide, lenalidomide, gemcitabine, thiotepa, fludarabine,carmustine, etoposide, cytarabine, granulocyte colony-stimulating factor(G-CSF), ADH-1, topotecan, palifermin, prednisone, arsenic trioxide,ascorbic acid, busulfan, cyclophosphamide,N,N′,N″-triethylenethiophosphoramide, buthionine sulfoximine, andcombinations thereof. A second therapeutic agent can be administered toa subject either in a pharmaceutical composition or unit dosage form ofthe present invention that includes at least one additional therapeuticagent (in addition to melphalan), or as a separate pharmaceuticalcomposition or unit dosage.

In some embodiments, the pharmaceutical compositions and/or unit dosageforms of the present invention are administered with other combinationsof therapeutic active agents such as, but not limited to, carmustine,etoposide and cytarabine; busulfan and thiotepa; doxorubicin andbortezomib; arsenic trioxide and citric acid; thalidomide and rituximab;thalidomide and prednisone; and busulfan, fludarabine and G-CSF.

In some embodiments, the pharmaceutical compositions and unit dosageforms of the present invention can enhance the bioavailability, rate oftherapeutic onset, and/or therapeutic efficacy of melphalan. Thus, thepresent invention is also directed to a method of decreasing the time totherapeutic onset of melphalan following administration thereof, themethod comprising orally or parenterally administering to a subject inneed thereof a pharmaceutical composition or unit dosage form of thepresent invention, wherein the time to therapeutic onset of melphalanprovided by the orally or parenterally administered composition or unitdosage is less than the time to therapeutic onset of melphalan providedby an orally administered reference composition that excludes thecyclodextrin derivative and contains an equivalent dose of melphalan. Insome embodiments, the time to therapeutic onset of melphalan followingadministration of a pharmaceutical composition or unit dosage form ofthe present invention is reduced by at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least50% compared to the time to therapeutic onset of melphalan provided byan intravenously administered reference composition that excludes thecyclodextrin derivative and contains an equivalent dose of melphalan.

In some embodiments, the dissolution of melphalan from the dosage formsof the present invention can be related to pharmacokinetic parametersand/or the in vivo concentration of melphalan and/or its metabolite(s).The in vivo concentration of melphalan and its metabolite(s), as well aspharmacokinetic parameters associated with an active form of melphalancan be determined by, e.g., sampling the blood plasma of a subject afteradministering a composition of the present invention. Pharmacokineticparameters that can be measured include, but are not limited to,AUC_(0-t), AUC_(t-∞), AUC_(0-∞), and ln(AUC_(LAST)).

As used herein, “AUC_(0-t)” refers to the Area Under the Concentrationtime curve (i.e., plot of plasma concentration vs. time) after melphalanadministration. The area is conveniently determined by the “trapezoidalrule”: the data points are connected by straight line segments,perpendiculars are erected from the abscissa to each data point, and thesum of the areas of the triangles and trapezoids so constructed iscomputed.

As used herein, “AUC_(t-∞)” refers to the Area Under the Concentrationtime curve, wherein the last concentration is extrapolated to baselinebased on the rate constant for elimination.

As used herein, “AUC_(0-∞)” refers to the sum of the Area Under theConcentration time curves for AUC_(0-t) and AUC_(t-∞).

As used herein, “In(AUC_(LAST))” refers to the Area Under theConcentration time curve determined by plotting plasma concentration ona natural logarithmic scale, using the last measured plasmaconcentration as the end point.

As used herein, “IntraCV” refers to an intra-assay coefficient ofvariation, which is the standard deviation within a sample set dividedby the mean value of the sample set, with the result reported as apercentage.

In some embodiments, the bioavailability of melphalan in a human subjectfrom a composition of the present invention is substantially greaterthan that observed upon administration of an equivalent dose ofmelphalan from a formulation lacking a cyclodextrin derivative (e.g.,ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable(Bioniche Pharma USA)). For example, the dosage forms of the presentinvention can have an AUC_(0-t) or AUC_(0-∞) that is at least 20% orgreater, at least 25% or greater, at least 30% or greater, at least 40%or greater, at least 50% or greater, at least 60% or greater, or atleast 70% or greater than the AUC_(0-t) or AUC_(0-∞) observed afteradministration of a melphalan formulation to a subject that contains thesame amount of melphalan and lacks a cyclodextrin derivative (e.g.,ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable(Bioniche Pharma USA)).

In some embodiments, the bioavailability of melphalan from a compositionof the present invention is greater than that observed uponadministration of an equivalent dose of melphalan from a formulationlacking a cyclodextrin derivative (e.g., ALKERAN® for Injection(GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).For example, the dosage forms of the present invention can have anAUC_(0-t) or AUC_(0-∞) that is at least 20% or greater, at least 25% orgreater, at least 30% or greater, at least 40% or greater, at least 50%or greater, at least 60% or greater, or at least 70% or greater than theAUC_(0-t) or AUC_(0-∞) observed after administration of a melphalanformulation to a subject that contains the same amount of melphalan andlacks a cyclodextrin derivative (e.g., ALKERAN® for Injection(GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)). Insome embodiments, the AUC_(0-t) or AUC_(0-∞) of melphalan from acomposition of the present invention is 20% to 70%, 20% to 60%, 20% to50%, 30% to 70%, 30% to 60%, 30% to 50%, 40% to 70%, 40% to 60%, or 50%to 70% greater than the AUC_(0-t) or AUC_(0-∞) observed uponadministration of an equivalent dose of melphalan from a formulationlacking a cyclodextrin derivative (e.g., ALKERAN® for Injection(GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).

In some embodiments, the maximum plasma concentration (C_(max)) ofmelphalan from a composition of the present invention is at least 20% orgreater, at least 25% or greater, at least 30% or greater, at least 40%or greater, at least 50% or greater, at least 60% or greater, or atleast 70% or greater than a C_(max) observed upon administration of anequivalent dose of melphalan from a formulation lacking a cyclodextrinderivative (e.g., ALKERAN® for Injection (GlaxoSmithKline) or MelphalanHCl Injectable (Bioniche Pharma USA)). In some embodiments, the maximumplasma concentration (C_(max)) of melphalan from a composition of thepresent invention is 20% to 70%, 20% to 60%, 20% to 50%, 30% to 70%, 30%to 60%, 30% to 50%, 40% to 70%, 40% to 60%, or 50% to 70% greater than aC_(max) observed upon administration of an equivalent dose of melphalanfrom a formulation lacking a cyclodextrin derivative (e.g., ALKERAN® forInjection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche PharmaUSA)).

In some embodiments, the rate of therapeutic onset of melphalan from acomposition of the present invention is faster than that observed uponadministration of an equivalent dose of melphalan from a formulationlacking a cyclodextrin derivative (e.g., ALKERAN® for Injection(GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche Pharma USA)).For example, the dosage forms of the present invention have a time toC_(max)(i.e., t_(max)) that is about 5%, about 10%, about 15%, about20%, about 25%, or about 30% faster, or about 35% faster than a t_(max)observed upon administration of an equivalent dose of melphalan from aformulation lacking a cyclodextrin derivative (e.g., ALKERAN® forInjection (GlaxoSmithKline) or Melphalan HCl Injectable (Bioniche PharmaUSA)). In some embodiments, the dosage forms of the present inventionhave a time to C_(max) (i.e., t_(max)) that is 5% to 35%, 5% to 30%, 5%to 25%, 5% to 20%, 10% to 35%, 15% to 35%, 20% to 35%, or 25% to 30%faster than a t_(max) observed upon administration of an equivalent doseof melphalan from a formulation lacking a cyclodextrin derivative (e.g.,ALKERAN® for Injection (GlaxoSmithKline) or Melphalan HCl Injectable(Bioniche Pharma USA)).

In some embodiments, the pharmaceutical compositions of the presentinvention provide a reduced rate of hypersensitivity in patients afterparenteral administration compared with patients parenterallyadministered a similar dose of melphalan without a cyclodextrinderivative.

In some embodiments, the pharmaceutical compositions of the presentinvention provide a reduced rate of severe myelotoxicity in patients(e.g., patients who experience a white blood cell count <1,000 per mLand/or platelet count <25,000) after parenteral administration comparedwith patients parenterally administered a similar dose of melphalanwithout a cyclodextrin derivative.

In some embodiments, the pharmaceutical compositions of the presentinvention provide a reduced rate of death in patients after parenteraladministration compared with patients parenterally administered asimilar dose of melphalan without a cyclodextrin derivative.

Having generally described the invention, a further understanding can beobtained by reference to the examples provided herein. These examplesare given for purposes of illustration only and are not intended to belimiting.

EXAMPLES Example 1

The dissolution rate of free-base melphalan (Chemwerth, Woodbridge,Conn.) in solutions at various pH and at various concentrations of acyclodextrin derivative were examined. The procedure was as follows:free base melphalan was added to a solution containing a cyclodextrinderivative (SBE_(6.5)-β-CD, CAPTISOL®) and then vortex mixed for 1-5minutes, and, if necessary, sonicated in ice water until a clearsolution was achieved.

TABLE Dissolution times for free base melphalan as a function ofcyclodextrin derivative concentration, volume, and pH. TargetDissolution Melphalan SBE_(6.5)-β-CD Time Conc. Conc. Volume pH (min) 50mg/mL 200 mM 5 mL 5 50 50 mg/mL 125 mM 6 mL 5 90 50 mg/mL 100 mM 7 mL 5160 50 mg/mL 75 mM 8 mL 5 >180 50 mg/mL 50 mM 10 mL  5 360 50 mg/mL 125mM 6 mL 2.7 75 50 mg/mL 125 mM 10 mL  1.8 16 50 mg/mL 125 mM 6 mL 1.3 550 mg/mL 75, 100 125 mM 10 mL  1.1 <5 for all

Referring to the data in the above Table, the dissolution of free basemelphalan was very rapid at pH 1.1 regardless of the concentration ofthe cyclodextrin derivative. After dissolution, the solution was thenneutralized with sodium hydroxide.

The solutions of free base melphalan in this example can be prepared byaddition of free base melphalan to a solution that contains thecyclodextrin derivative, or by adding a 0.1 M HCl solution to themelphalan and then adding the cyclodextrin derivative, or dissolving thefree base melphalan and cyclodextrin derivative simultaneously. However,sonication was superior to mixing and/or shaking for dissolutionenhancement and de-clumping of dry material in solution.

Example 2

The binding of free-base melphalan (Chemwerth, Woodbridge, Conn.) wasstudied as a function of cyclodextrin derivative concentration at pH 5and pH 7, and the data was compared with literature reports of free-basemelphalan binding. The pH 5 solutions contained 100 mM sodium bitartratebuffer, and was adjusted to pH 5 using sodium hydroxide in 0.9% sodiumchloride solution. The pH 7 solutions contained 50 mM each of mono- anddi-basic phosphate and 0.9% sodium chloride. Solutions containing 0, 50,75 and 100 mM SBE_(6.5)-β-CD (CAPTISOL®) were prepared, and excess freebase melphalan was added to 2 mL samples of each solution. After theaddition of free base melphalan, the samples were vortex mixed for 30seconds, sonicated in ice water bath for 20 minutes, and then mixed byend-over-end rotation at room temperature for 30 minutes. The sampleswere then centrifuged, the clear supernatant was diluted with water, andanalyzed by HPLC.

All melphalan assays performed by HPLC utilized the following protocol.A Shimadzu HPLC equipped with a SCL-10A system controller, SIL-10A autoinjector, LC-10AT liquid chromatograph, SPD-10A UV spectrophotometerdetector, CTO-10A column oven, and Class-VP chromatography laboratoryautomated software was utilized. The column was a ZORBAX® RX-C18 4.6 mmby 150 mm column (Agilent Technologies) having a 5 μm particle size.Samples were injected (20 μL) onto the column for isocratic elutionusing mobile phase of phosphate buffered saline (pH7.4):methanol:glacial acetic acid in a ratio of 500:250:10 (v/v). Themobile phase was selected in order to decrease or nominally quenchmelphalan conversion by having a high-chloride concentration. Thesamples were prepared immediately prior to injection. Detection was at260 nm.

The literature procedure involved adding an excess amount of melphalanto 0, 10, 20, 30, 40, 50, 75 and 100 mM solutions of SBE₇-β-CD (avg.M.W.=2248 g/mol) in a 25 mM phosphate buffer solution at pH 7.5. Thesuspensions were placed in tightly capped vials, sonicated 1 h, andagitated at 25° C. for 23 h. The solutions were then centrifuged, theclear supernatant was diluted with doubly distilled water, and analyzedby HPLC. See D. Q. Ma et al., J. Pharm. Sci. 89:275 (1999).

The data from the free base melphalan solubilization studies areprovided in FIG. 1. Referring to FIG. 1, free base melphalan displayed asignificantly lower solubility enhancement than that provided in aprevious literature report. See id. Because the solubility enhancementof free base melphalan provided by SBE_(6.5)-β-CD was lower thanexpected, additional phase solubility tests were performed usingmelphalan hydrochloride.

Example 3

The binding of melphalan hydrochloride (USP reference standard) and freebase melphalan (Chemwerth) with a cyclodextrin derivative(SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol) was determined as afunction of cyclodextrin derivative concentration at pH 7.5. Thetemperature was maintained at 22° C., and a 25 mM phosphate buffer wasadded to each solution. The data was compared with a literature reportof free-base melphalan binding with SBE₇-β-CD (avg. M.W.=2248 g/mol) ina 25 mM phosphate buffer at pH 7.5 (see Example 2).

The samples were prepared by adding excess melphalan hydrochloride orfree base melphalan to a 1 mL sample of various SBE_(6.5)-β-CDsolutions. The samples were vortex mixed for 30 seconds, sonicated at20-24° C. for 60 minutes, and then mixed by end-over-end rotation at 22°C. for 60 minutes. The samples were then centrifuged, the clearsupernatant was diluted with water, and analyzed by HPLC. The data areprovided in FIG. 2. Referring to FIG. 2, the melphalan hydrochloridesalt displayed a significant solubility enhancement compared to freebase melphalan for all cyclodextrin derivative concentrations above 25mM.

Example 4

A pharmaceutical composition comprising melphalan as a hydrochloridesalt was prepared by the process outlined schematically in FIG. 3.Referring to FIG. 3, water for injection, USP was placed in a stainlesssteel mixer at a temperature of 15-20° C., and hydrochloric acid wasadded until a pH of about 4.6 was achieved. The resulting solution wasstirred at a speed sufficient to produce a vortex (but without foamingor frothing) for about 15 minutes, a cyclodextrin derivative (27.2 gSBE_(6.5)-β-CD, CAPTISOL®) was added slowly while vortex stirring, andthe resulting solution was stirred for about 15 minutes to ensurecomplete dissolution. The resulting solution had a pH of about 2.5.Melphalan as a hydrochloride salt (516 mg) was added slowly while vortexstirring, and the resulting solution was stirred for about 15 minutes toensure complete dissolution. A base (2 N NaOH) was then slowly addedwhile vortex stirring until the solution had a pH of about 5.6. Thesolution was then assayed using a UV/vis spectrophotometer (detectionwavelength of 260 nm). The solution comprised melphalan at aconcentration of 5.16 mg/mL, and the melphalan was present in a ratio ofabout 1:55 w/w relative to the cyclodextrin derivative. The solution wasthen passed through a sterile filter (0.22 μm PVDF) and cooled to 10-15°C.

Example 5

The liquid pharmaceutical composition provided in Example 4 waslyophilized to provide a reconstitutable and/or dilutable dry powdercomprising 50 mg of melphalan as a hydrochloride salt. Glass vials werefilled with the solution (10 mL) and placed in trays on a pre-cooledshelf at 5° C. The vials were allowed to thermally equilibrate for about30 minutes, and were then lyophilized to provide a dry powder in eachvial. The vials were back-filled with nitrogen at a pressure of about400 mTorr, and then sealed.

Example 6

A pharmaceutical composition comprising melphalan as a hydrochloridesalt was prepared by the process described in Example 4, and outlinedschematically in FIG. 3, except that the final solution containedmelphalan at a concentration of 10 mg/mL, and the melphalan was presentin a ratio of about 1:27 w/w relative to the cyclodextrin derivative.

Example 7

The liquid pharmaceutical composition provided in Example 6 waslyophilized to provide a reconstitutable and/or dilutable dry powdercomprising 200 mg of melphalan as a hydrochloride salt. Glass vials werefilled with the solution (20 mL) and placed in trays on a pre-cooledshelf at 5° C. The vials were allowed to thermally equilibrate for about30 minutes, and were then lyophilized to provide a dry powder in eachvial. The vials were back-filled with nitrogen at a pressure of about400 mTorr, and then sealed, packaged, and labeled. The vials wereprotected from exposure to light during all aspects of thelyophilization, back-filling, sealing, packaging and labelingprocedures.

Prophetic Example A

The liquid pharmaceutical composition provided in Examples 4 and 6 willbe aseptically spray dried to provide a free-flowing powder to be filledaseptically. The free-flowing powder will meet or exceed the dissolutionproperties of the lyophilized powder prepared in Examples 4 or 6.

Example 8

A pharmaceutical composition comprising melphalan as a hydrochloridesalt was prepared by the process outlined schematically in FIG. 4.Referring to FIG. 4, water for injection, USP was placed in a stainlesssteel mixer at a temperature of 18-25° C., and the resulting solutionwas stirred at a speed sufficient to produce a vortex (but withoutfoaming or frothing). A cyclodextrin derivative (SBE_(6.5)-β-CD,CAPTISOL®) was added slowly while vortex stirring, and the resultingsolution was stirred for about 15 minutes to ensure completedissolution. The resulting solution was then cooled to about 2-8° C.Melphalan as a hydrochloride salt was added slowly while vortexstirring, and the resulting solution was stirred for about 15 minutes toensure complete dissolution. A base (2 N NaOH) was then slowly addedwhile vortex stirring until the solution had a pH of about 5-6 (targetpH 5.5). An in-process control (“IPC”) assay was then performed tomonitor pH, and the solution was diluted to the final target volumeusing water for injection, USP. The solution was then assayed using aUV/vis spectrophotometer (detection wavelength of 260 nm) and abioburden assay was performed. The solution was then passed through asterile filter (0.22 μm PVDF) and cooled to 15-25° C. Finally, an IPCassay was conducted.

Example 9

The solution prepared in Example 8 was lyophilized to provide areconstitutable and/or dilutable dry powder comprising melphalan as ahydrochloride salt. For the lyophilization, glass vials were filled withthe solution (10 mL) and placed in trays on a pre-cooled shelf at 5° C.The vials were allowed to thermally equilibrate for about 1 hour, andwere lyophilized to provide a dry powder in each vial. The vials wereback-filled with nitrogen, sealed, packaged, and labeled. The vials wereprotected from exposure to light during all aspects of thelyophilization, back-filling, sealing, packaging and labelingprocedures.

Prophetic Example B

The liquid pharmaceutical composition provided in Example 8 will beaseptically spray dried to provide a free-flowing powder to be filledaseptically. The free-flowing powder will meet or exceed the dissolutionproperties of the lyophilized powder prepared in Example 9.

Example 10

The properties of the pharmaceutical compositions of the presentinvention after dilution with Water for Injection, USP, were analyzed bya variety of analytical methods. The results are listed in the Tablebelow. Compositions A-D were prepared by the process described inExamples 8-9. The diluted compositions contained SBE_(6.5)-β-CD(CAPTISOL®) in a concentration of 75 mM, 100 mM, 125 mM, and 125 mM,respectively. Each of the compositions had a moisture content of about1.3% to about 2.5% prior to dilution.

TABLE Properties of pharmaceutical compositions of the present inventioncontaining varying concentrations of a cyclodextrin derivative. DilutionSBE_(6.5)-β-CD Dissolution Density Time for Volume Conc. Time pH (22°C.) Viscosity 5% loss A 10 mL  75 mM <30 s 5.05 1.07 g/cc 2.06 cP 10 h B10 mL 100 mM <30 s 4.9 1.08 g/cc 2.28 cP 23 h C 10 mL 125 mM  45 s 5.051.11 g/cc 2.95 cP 49 h D  5 mL 125 mM 105 s 5.2 1.11 g/cc 3.02 cP 25 h

Example 11

The stability of melphalan hydrochloride upon dilution of apharmaceutical composition of the present invention was determined.Pharmaceutical compositions containing a cyclodextrin derivative(SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol) were diluted withisotonic saline to provide 0.45 mg/mL melphalan solutions that containedthe cyclodextrin derivative at a concentration of 75 mM and 125 mM,respectively. Melphalan was assayed as a function of time, and the timenecessary for a 5% or 10% loss of melphalan (based on an initialmelphalan concentration of 100%) was determined. The data is provided inthe Table below. The times required for melphalan to fall to 90% or 95%of its initial concentration in the solutions that contained thecyclodextrin derivative were compared to the stability of melphalan in aReference product (ALKERAN® for Injection, GlaxoSmithKline).

TABLE Melphalan stability as a function of cyclodextrin derivativeconcentration, compared to a reference melphalan standard. Time for Timefor 5% loss 10% loss SBE_(6.5)-β-CD (75 mM) 5.4 h 11 h SBE_(6.5)-β-CD(125 mM) 8.8 h 18 h Reference 1.3 h 2.7 h 

Referring to the data in the above Table, the stability of melphalanafter dilution from a pharmaceutical composition of the presentinvention shows an improvement of approximately 4.2 times and 6.8 timesat a cyclodextrin derivative concentration of 75 mM and 125 mM,respectively, compared to a reference melphalan formulation that doesnot contain a cyclodextrin derivative.

Example 12

The stability of melphalan hydrochloride upon dilution of apharmaceutical composition of the present invention was determined as afunction of temperature and storage conditions. Pharmaceuticalcompositions containing melphalan (50 mg) and a cyclodextrin derivative(SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol, 270 mg) were dilutedwith isotonic saline (8.5 mL) to provide a concentrated solution. Theconcentrated solution was further diluted 10-fold to provided a dilutesolution. Each of the concentrated and diluted melphalan solutions werestored at 25° C./60% relative humidity, or in a refrigerator (˜10° C.),and the melphalan content was monitored as a function of time. The datais provided in the Table below.

TABLE Melphalan stability as a function of temperature and storageconditions. Storage Melphalan Monohydroxy Solution Time Conditions AssayMelphalan Assay Conc. Sol'n 0 Refrigerator 99% 0.8% ″ 6.5 ″ 98.9%   0.8%″ 24.5 ″ 98.8%   0.9% ″ 48.5 ″ 98.4%     1% Conc. Sol'n 0 25° C./60%R.H. 99% 0.8% ″ 6 ″ 98% 1.5% ″ 24 ″ 96% 3.4% ″ 48 ″ 93% 5.7% DiluteSol'n 0 Refrigerator 99%   1% ″ 5 ″ 98.4%   1.4% ″ 24.3 ″ 97.8%     2% ″48.4 ″ 96.7%   2.7% Dilute Sol'n 0 25° C./60% R.H. 99%   1% ″ 5.3 ″ 94%5.3% ″ 23.8 ″ 81% 15.5%  ″ 47.2 ″ 70% 20.2% 

Referring to the data in the above Table, the stability of melphalanafter dilution from a pharmaceutical composition of the presentinvention provides a significant improvement compared to currentlyavailable melphalan pharmaceutical compositions that do not contain acyclodextrin derivative.

Example 13

The stability of a lyophilized melphalan hydrochloride composition wasdetermined before and after dilution as a function of temperature andstorage conditions. The study was performed under the direction of CyDexPharmaceuticals, Inc. by BioConvergence LLC, Bloomington, Ind.

Compositions comprising melphalan (50 mg) and povidone (20 mg) werediluted with compositions comprising a cyclodextrin derivative(SBE_(6.5)-β-CD, CAPTISOL®, avg. M.W.=2163 g/mol, 270 mg), sodiumcitrate (200 mg), and distilled water (10 mL) to provide concentratedmelphalan solutions (5 mg/mL). In addition to testing the stability ofthe concentrated solutions, further dilution 11-fold to provide dilutesolutions containing melphalan (0.45 mg/mL).

The kinetic stability of a reconstituted concentrated solution (5 mg/mLmelphalan) was determined upon storage in a glass vial, and the kineticstability of a reconstituted diluted solution (0.45 mg/mL melphalan) wasdetermined upon storage in a 50 mL Baxter INTRAVIA® bag, at refrigerated(about 2°-8° C.) and room (about 25° C., monitored under fluorescentlight) temperatures.

Evaluation of the kinetic stability of the dilute (0.45mg/ml melphalan)composition was determined in a 50 mL Type I glass container:reconstitution was performed using saline (8.5 mL), and aliquots (4.5mL) were removed from each vial and injected into 4 glass containersthat held 45.5 mL of saline. After an amount was withdrawn from eachcontainer for t=0 analysis, the containers were stored at roomtemperature (“RT”, about 25° C., under fluorescent light), or in arefrigerator (about 2°-8° C.), and the melphalan content was monitoredas a function of time. The data is provided in the Table below.

TABLE Summary of melphalan stability as a function of temperature andstorage conditions Hold MEL Degradation Run Form Storage Conditions Time(% w/w) 1 Lyophilized comp. RT (~20°-25° C.)  2 yrs <2% Conc. solution(5 mg/mL) Immediately diluted n/a n/a Dilute solution (0.45 mg/mL) RT(~20°-25° C.) 10 h <4% 2 Lyophilized comp. RT (~20°-25° C.)  2 yrs <2%Conc. solution (5 mg/mL) RT (~20°-25° C.) 24 h <4% Dilute solution (0.45mg/mL) RT (~20°-25° C.)  5 h <2% 3 Lyophilized comp. RT (~20°-25° C.)  2yrs <2% Conc. solution (5 mg/mL) Refrigerated (2°-8° C.) 48 h <4% Dilutesolution (0.45 mg/mL) RT (~20°-25° C.)  5 h <2% 4 Lyophilized comp. RT(~20°-25° C.)  2 yrs <2% Conc. solution (5 mg/mL) Immediately dilutedn/a n/a Dilute solution (0.45 mg/mL) 1) Refrigerated (2°-8° C.) 1) 24h 1) <2% 2) RT (~20°-25° C.) 2) 5 h 2) <2%

Referring to the data in the above Table, the stability of melphalanafter dilution from a pharmaceutical composition of the presentinvention provides a stable composition that can be maintained at roomtemperature for up to 5 hours and exhibit less than 2% melphalandegradation, or up to 10 hours and exhibit less than 4% melphalandegradation. When refrigerated, a diluted melphalan composition can bestored up to 24 hours and exhibit less than 2% melphalan degradation.Additionally, a lyophilized pharmaceutical composition can be stored upto 2 years at room temperature and exhibit less than 2% melphalandegradation.

Furthermore, referring to Examples 12 and 13, the pharmaceuticalcompositions of the present invention provide a significant improvementin melphalan stability compared to other formulations in which the useof a cyclodextrin derivative has been proposed. For example, D. Q. Ma etal., Int. J. Pharm. 189:227 (1999) provide a melphalan composition thatupon dilution with a solution containing a cyclodextrin derivative,exhibits a melphalan loss of more than 60% after 48 hours at roomtemperature. Significantly, the data in the above Tables illustratesthat upon dilution of a pharmaceutical composition of the presentinvention, a melphalan loss of at most 30% is observed within 48 hoursat room temperature. The melphalan loss can be reduced to as low as 2%or 3% when the solution is stored at a reduced temperature (e.g., in arefrigerator).

Example 14

The hemolytic potential of a cyclodextrin derivative suitable for usewith the pharmaceutical composition of the present invention wasanalyzed in comparison to a previously marketed diluent vehicle formelphalan (ALKERAN® For Injection, GlaxoSmithKline). The hemolyticpotential was evaluated in rodent (SPRAGUE DAWLEY® or Wistar Han IGSrats) and human red blood cells obtained from fasted subjects using aspectrophotometric technique. Normal saline (0.9% sodium chloride) wasused as the blank (or background) and as a negative control forcomparison against various concentrations of a cyclodextrinderivative-containing, and cyclodextrin-free diluent vehicles. Apositive control containing Triton X-100 (1%) in phosphate bufferedsaline was also utilized. Human red blood cells were taken from fasted(≧8 h) adult subjects. The components of the various samples are listedin the following Table.

TABLE Components of diluent vehicles used for hemolysis studies.Identification Constituents Negative Control/Blank 0.9% Sodium ChloridePositive Control 1% TRITON ® X-100 in phosphate buffered salineCyclodextrin Derivative SBE_(6.5)-β-CD (CAPTISOL ®, 9.72 g) Diluent q.s.400 mL with normal saline ALKERAN ® for Injection Povidone (K-12, 72 mg)(GlaxoSmithKline) Diluent Sodium citrate (720 mg) Propylene glycol (21.6mL, 22.4 g) Ethanol (1.87 mL, 1.48 g) Water (12.2 mL) q.s. 400 mL withnormal saline

The rat and human red blood cells were exposed to various concentrationsof the diluent vehicles and the hemolytic potential was evaluated usingequation (1):

A ₅₄₀(test article)−A ₅₄₀(negative control)×100=% Hemolysis   (1)

The hemolysis results are provided in the following Table, where Group Arefers to rat red blood cells exposed to the cyclodextrin derivativevehicle; Group B refers to rat red blood cells exposed to the ALKERAN®for Injection (GlaxoSmithKline) diluent vehicle; Group C refers to humanred blood cells exposed to the cyclodextrin derivative vehicle; andGroup D refers to human red blood cells exposed to the ALKERAN® forInjection (GlaxoSmithKline) diluent vehicle. The negative controls foreach experiment provided absorbances below 0.13, and the positivecontrols for each experiment provided absorbances of about 2.8 to 3.

TABLE Hemolysis results for rat and human red blood cells exposed tovarious diluent vehicles. Dilution Group Meas. none 1:2 1:4 1:8 1:161:32 1:64 1:128 A Abs. (a.u.) 0.11 0.114 0.11 0.117 0.116 0.118 0.1190.118 Hem. (%) 1% 0 0 0 0 0 0 0 B Abs. (a.u.) 0.178 0.155 0.141 0.1280.125 0.121 0.123 0.124 Hem. (%) 2% 1% 1% 0 0 0 0 0 C Abs. (a.u.) 0.0210.021 0.022 0.019 0.017 0.016 0.017 0.025 Hem. (%) 0 0 0 0 0 0 0 0 DAbs. (a.u.) 0.037 0.031 0.033 0.02 0.025 0.022 0.027 0.027 Hem. (%) 0 00 0 0 0 0 0

Referring to the hemolysis data in the above Table, the solution thatcontained the cyclodextrin derivative at high concentrations (e.g., nodilution, 1:2 dilution with saline, and 1:4 dilution with saline)provided reduced hemolysis in rat red blood cells, which was alsoexhibited as a reduction in spectrophotometric absorption of about 30%compared to the ALKERAN® for Injection (GlaxoSmithKline) diluent. Whilethe hemolysis tests in human blood cells exhibited a similar reductionin spectrophotometric absorbance at high concentrations, neither thecyclodextrin derivative solution or the ALKERAN® for Injection(GlaxoSmithKline) diluent vehicle induced hemolysis in human red bloodcells.

Example 15

A study was conducted that determined melphalan associated withSAE_(6.5)-β-CD (CAPTISOL®, CyDex Pharmaceuticals, Inc., Lenexa, Kans.)exhibits the same protein binding as unassociated melphalan. The studywas performed under the direction of CyDex Pharmaceuticals, Inc. byAnalytical Biochemistry Laboratories, Inc., Columbia, Mo.

Preliminary Study

A preliminary study was performed that determined radioactive-labeledmelphalan, [¹⁴C]-melphalan (Moravek Biochemicals, Inc., Brea, Calif.),does not bind non-specifically to ultrafiltration devices. The followingmixtures of compounds were added to human plasma ultrafiltrate(Biochemed, Winchester, Va.) to determine the protein binding of[¹⁴C]-melphalan alone or in combination with SAE_(6.5)-β-CD:

-   -   1. [¹⁴C]-melphalan with melphalan; and    -   2. [¹⁴C]-melphalan with melphalan and SAE_(6.5)-β-CD.

Radioactive-labeled warfarin, [³H]-warfarin (Moravek Biochemicals, Inc.,Brea, Calif.), a compound with well-documented protein bindingproperties was used as a positive control in all experiments.

Powdered material (as applicable) was weighed into scintillation vials(20 mL) and the radiolabeled compounds were added to the vials using apositive displacement pipette. Blank human plasma ultrafiltrate (5 mL)was then added to the vials using a glass serological pipette. Themixtures were then blended briefly. The time-dependence of plasmaprotein binding was determined by sampling the mixtures 0.5, 1, and 5minutes after addition of the test compounds to the human plasmaultrafiltrate. The sample aliquots (3×1 mL) were dispensed into theultrafiltration devices, and the samples were immediately centrifuged(1600 g for 5 minutes at 25° C.). The solution remaining in the vialswas then aliquoted for Liquid Scintillation Counting (LSC) analysis(2×0.1 mL).

More than 95% recovery was observed for [³H]-warfarin in allexperiments. In the protein-binding experiments, [³H]-warfarin was over99% protein bound. Radiolabeled melphalan (alone or in combination withSAE_(6.5)-β-CD) applied to ultrafiltration devices having a molecularweight cutoff of 30 kD exhibited an average of over 97% radioactivityrecovery: [¹⁴C]-melphalan alone exhibited a recovery of 97.7% (n=3), and[¹⁴C]-melphalan with SAE_(6.5)-β-CD exhibited a recovery of 97.6% (n=3).The results demonstrate that there was minimal (i.e., less than 2.4%)non-specific binding of radiolabeled melphalan to the ultrafiltrationdevices.

Protein-Binding Study

For the protein-binding study, the radiolabeled melphalan,non-radiolabeled melphalan, and SAE_(6.5)-β-CD (as applicable) wereadded to a scintillation vial (20 mL) and blown to dryness under anitrogen stream to standardize the amount of solvent present in eachexperiment. Methanol (50 μL) was added to the vials and blank humanplasma (5 mL) was added to the vials using a glass serological pipette.The mixtures were then blended briefly. Aliquots (3×1 mL) were thendispensed from the vials into the ultrafiltration devices, followed bycentrifugation (2,000 g for 5 minutes at 25° C.). The time intervalbetween adding human plasma to the vials and the start of centrifugationwas 0.5, 1, 5, 10, and 30 minutes. The solution remaining in the vialswas then aliquoted in duplicate (at a volume of 0.1, 0.05, and 0.025 mL)for LSC analysis.

Sample radioactivity was quantified using a scintillation counter(Beckman Instruments, Inc. Schaumberg, Ill.) equipped with the H-numbermethod for cpm to dpm conversion. LSC analysis was performed withsamples (5 mL) in glass scintiallation vials (7 mL), from whichbackground measurements were made using the same amount of scintillationfluid added to the vials. The results are provided in the followingTable:

TABLE Protein binding of radiolabeled melphalan (“[¹⁴C]-mel”) withunlabeled melphalan (“mel”) in the presence and absence ofSAE_(6.5)-β-CD. Total Conc. Conc. Recovery (%) % Binding Mixture(dpm/mL) (μg/mL) Rep. Data Avg. Data Avg. [¹⁴C]-mel + mel 112,105 14 196.4 97.1 64.5 64.3 ± 0.34 (1 min.) 2 96.9 63.9 3 98.0 64.4 [¹⁴C]-mel +mel + 112,382 14 1 92.9 91.5 63.7 64.3 ± 0.69 SAE_(6.5)-β-CD 2 95.4 64.1(0.5 min.) 3 86.4 65.1 [¹⁴C]-mel + mel + 108,085 13 1 97.3 97.5 63.864.4 ± 0.59 SAE_(6.5)-β-CD (1 min.) 2 97.0 64.8 3 98.3 64.6 [¹⁴C]-mel +mel + 109,621 13 1 96.5 95.9 65.8 67.2 ± 1.96 SAE_(6.5)-β-CD (5 min.) 295.7 69.5 3 96.5 66.4 [³H]-warfarin 114,932 0.052 1 98.5 101 98.9 98.8 ±0.01 2 100 98.8 3 103 98.8

The results showed that [¹⁴C]-melphalan in the absence of SAE_(6.5)-β-CDwas 64.3% protein bound after 1 minute in human plasma. Similar degreesof protein binding were observed for [¹⁴C]-melphalan in the presence ofSAE_(6.5)-β-CD: 64.3% (0.5 minutes, n=3), 64.4% (1 minute, n=3), and67.2% (5 minutes, n=3). The study showed that SAE_(6.5)-β-CD did notaffect the protein-binding of radiolabeled [¹⁴C]-melphalan.

Example 16

A study was performed to investigate the potential for a sulfoalkylether cyclodextrin to perturb the in vivo pharmacokinetics of melphalan.Pharmacokinetic parameters were determined for melphalan followingintravenous administration to male Sprague Dawley rats in the presenceor absence of SBE_(6.5)-β-CD in the delivery vehicle.

The pharmacokinetics of melphalan were studied in overnight-fasted maleSprague Dawley rats. All experimental procedures were approved andperformed in accordance with the guidelines of the Institutional AnimalExperimentation Ethics Committee (Monash University Ethics approvalnumber VCPA/2008/02).

On the day prior to dosing, a commercially available BASi CULEX® cannula(for use with a CULEX® automated blood sampling device) was insertedinto the left carotid artery of each rat under isoflurane anesthesia(2%). A polyethylene cannula was also inserted into the right jugularvein. Cannulae were exteriorized by tunneling subcutaneously to emergeabove the scapulae.

Immediately following surgery and through to the end of the experiment,rats were housed in RATURN® metabolic cages in the CULEX® automatedblood sampler. All rats returned to normal grooming, drinking andsleeping behavior within an hour of surgery. Animals were given a smallamount of food just after they awoke from the anesthetic, but were thenfasted for 16-18 hours prior to drug administration. Animals had accessto water ad libitum at all times. Food was reinstated 4 hours followingdrug administration. At the conclusion of each experiment, rats werekilled by a single lethal injection of pentobarbitone.

The cyclodextrin derivative-free melphalan formulation was prepared asper the product insert for ALKERAN® for Injection (GlaxoSmithKline). Thecontents of a single ALKERAN® for Injection (GlaxoSmithKline) vial werereconstituted with 10 mL of sterile diluent (provided with the ALKERAN®product, and containing 0.2 g sodium citrate, 6 mL propylene glycol,0.52 mL ethanol (96%) and water). The solution was then further dilutedwith 0.9% normal saline (2 mL ALKERAN® for Injection (GlaxoSmithKline)in 10 mL of 0.9% saline, i.e., 12 mL total volume), and the resultantformulation was sterilized by filtering through a 0.22 μm syringe filterbefore administering to rats. The measured concentration of melphalan inthe IV formulation was 0.54 mg/mL (as free base) and the pH of the finalsolution was between 5 and 6 (checked using pH paper). The formulationwas administered to animals within 30 minutes of preparation.

A formulation containing SBE_(6.5)-β-CD (27% w/v) was prepared bydissolving SBE_(6.5)-β-CD in Milli-Q water. Contents of an ALKERAN® forInjection (GlaxoSmithKline) vial were then reconstituted with 10 mL ofthe SBE_(6.5)-β-CD solution. This solution was then diluted with 0.9%saline (2 mL in 10 mL of 0.9% saline) and the resultant formulation wassterilized by filtering through a 0.22 μm syringe filter beforeadministering to rats. Thus, the final formulation contained 4.5% (w/v)SBE_(6.5)-β-CD. The measured concentration of melphalan (as free base)was 0.58 mg/mL and the pH of the final solution was between 5 and 6(checked using pH paper). The formulation was administered to animalswithin 30 minutes of preparation.

The total dose volume was 1 mL and all doses were infused manually viathe jugular vein cannula. The complete dose was delivered over a10-minute period and the cannula was flushed with heparinized saline (10U/mL) to ensure complete administration of the dose.

Samples of arterial blood and urine were collected according to thefollowing schedules: blood/plasma sampling times were pre-dose, and 5,10 (end of infusion), 15, 25, 40, 55, 70, 100, 130, 190, 250, 370, and490 minutes post-dose; and urine sampling intervals were 0-70 minutes,70-130 minutes, 130-190 minutes, 190-250 minutes, 250-310 minutes,310-370 minutes, 370-430 minutes, 430-490 minutes, and 490-1450 minutes.

Arterial blood was collected directly into borosilicate vials (at 4° C.)containing heparin, COMPLETE® (a protease inhibitor cocktail), potassiumfluoride, and EDTA to minimize potential for ex vivo degradation inblood/plasma samples. Once collected, an aliquot (50 μL) of whole bloodwas transferred into a fresh micro-centrifuge tube. The remaining bloodwas centrifuged and supernatant plasma removed. All blood, plasma andurine samples were immediately (snap) frozen on dry ice and thentransferred to −20° C. freezer for storage until analysis.

The melphalan concentration in whole blood, plasma, urine and samples ofdosing solutions were determined using LC-MS.

Sample preparation was performed using protein precipitation withacetonitrile. Aliquots of the plasma and blood (50 μL) were treated withinternal standard (10 μL), acetonitrile (130 μL), vortexed andcentrifuged. The supernatant was removed and analyzed by LC/MS. Standardsamples were prepared by spiking solution standards in the respectiveblank matrix. A stock solution of melphalan free base was prepared at aconcentration of 10 mg/mL in dimethylsulfoxide. This stock solution wasfurther diluted in aqueous acetonitrile (50% v/v) to obtain spikingsolutions for the preparation of calibration standards.

All samples were assayed via LC-MS/MS on a Micromass Quattro Premier PRtriple quadrupole instrument coupled with a Waters Acquity UPLC (WatersCorp., Milford, Mass.). Analytical separations were performed on aPhenomenex Polar reverse-phase column (50 mm×1.0 mm inner diameter, 4 μmparticle size) equipped with a Phenomenex Polar Security Guard column ofthe same material (Torrance, Calif.). Samples (7.5 μL) were injectedonto the column and compounds were eluted (at a flow rate of 0.15mL/min) using a ternary gradient solvent system consisting of an aqueoussolution of methanol (2% v/v) and formic acid (0.05% v/v) in Milli-Qwater (solvent A), and acetonitrile containing formic acid (0.05% v/v)(solvent B). The gradient conditions used for LC-MS analysis are listedin the following table.

TABLE Gradient chromatography conditions used for the analysis ofmelphalan. Time (minutes) Solvent A Solvent B   0-0.2 89 2 0.3 80 20 2.720 80 2.8 5 95 3.3 5 95 3.5-4.5 98 2

Elution of analytes was confirmed by multiple reaction monitoring (MRM)using diazepam (0.2 μg/mL) as the internal standard (diazepam). Inletcone voltages were 20 eV and 40 eV for melphalan and the internalstandard, respectively, and collision energies of 15 eV and 27 eV formelphalan and the internal standard, respectively. The elution ofmelphalan and internal standard was monitored using the followingtransitions 304.94>267.88 and 285.17>154.02, respectively. Melphalanexhibited a retention time of 2.0 minutes and the internal standardexhibited a retention time of 2.8 minutes.

Mass spectrometry was performed using positive mode electrosprayionization with a capillary voltage of 3.2 kV, detector multiplier gainof 650 V, and source block and desolvation temperatures of 90° C. and300° C., respectively. A desolvation gas (nitrogen) and collision gas(argon) flow of 500 L/h and 0.38 mL/min, respectively, was maintained.The lower limit of quantitation (LLQ) for blood and plasma standards was5.0 ng/mL, and the LLQ for diluted urine samples was 0.5 ng/mL.

Both plasma and blood concentration data were analysed to obtainpharmacokinetic parameters using WINNONLIN® software (WINNONLIN®professional version 5.2.1, Pharsight Corp., Mountain View, Calif.). Thetotal clearance (CL_(total), for whole blood or plasma) afterintravenous administration was calculated as: CL_(total)=Dose/AUC, whereAUC is the area under the whole blood or plasma concentration versustime curve obtained using the linear trapezoidal method. The volume ofdistribution (V_(z)) was calculated as: V_(z)=CL_(total)/λ_(z), whereλ_(z) is the elimination rate constant after i.v. administration.

The mean dose-normalized concentration versus time profiles of melphalanin whole blood and plasma following intravenous administration using aformulation containing SBE_(6.5)-β-CD (n=4) and a cyclodextrin-free(n=5) formulation are presented in the following table.

TABLE Pharmacokinetic parameters for melphalan in whole blood and plasmafollowing intravenous administration to male Sprague Dawley rats at anominal dose of 2.0 mg/kg with a formulation containing a cyclodextrinderivative (SBE_(6.5)-β-CD) and a formulation free from a cyclodextrinderivative (“CD-free formulation”). Whole Blood Plasma SBE_(6.5)-β-CDCD-free SBE_(6.5)-β-CD CD-free Melphalan (27% w/v) formulation (27% w/v)formulation Apparent t_(1/2) (h) 0.8 ± 0.1 0.9 ± 0.1 0.8 ± 0.1 0.9 ± 0.1CL_(total) (mL/min/kg) 10.9 ± 2.0  12.2 ± 1.3  8.1 ± 1.1 9.3 ± 1.4 V_(z)(L/kg) 0.8 ± 0.2 1.0 ± 0.2 0.6 ± 0.1 0.7 ± 0.2 AUC_(0-inf)/D (μM · min ·kg/μmol) 94.2 ± 16.6 82.8 ± 8.8  125.4 ± 15.0  109.6 ± 16.1 

FIGS. 5A-5B provide graphic representations of the dose-normalized wholeblood (FIG. 5A) and plasma (FIG. 5B) concentrations of melphalanfollowing intravenous administration for the SBE_(6.5)-β-CD-containingmelphalan formulation (), n=4, and the cyclodextrin-free (∘), n=5,melphalan formulation (ALKERAN® for Injection, GlaxoSmithKline).Referring to FIGS. 5A-5B, the data is presented as mean with error barsindicating a single standard deviation. Melphalan exhibitedbi-exponential pharmacokinetics in both whole blood and plasma and theapparent terminal elimination phase was well-defined within the 8 hourpost-dose blood sampling period, with the apparent terminal eliminationhalf-life. The mean whole blood and plasma concentration versus timeprofiles for the formulation that contained SBE_(6.5)-β-CD (27% w/v) andcyclodextrin-free formulation were essentially super-imposable and therewere no statistically significant differences in any of thepharmacokinetic parameters between the two formulations (p>0.05). Thus,as shown in the above table, in the rat the in vivo pharmacokineticparameters for a melphalan formulation containing SBE_(6.5)-β-CD areessentially identical to the pharmacokinetic parameters for acyclodextrin-free formulation (i.e., ALKERAN® for Injection,GlaxoSmithKline).

Additionally, in both formulations the percentage of the melphalan doseexcreted in urine excreted as unchanged compound up to 24 hourspost-dose was low: for the cyclodextrin-free formulation the average was2.7±1.7%, and for the formulation containing SBE_(6.5)-β-CD the averagewas 2.3±2%.

The data show that pharmacokinetic parameters, including half-life, AUC,volume of distribution, clearance, and the extent of renal eliminationof melphalan were essentially unchanged between ALKERAN® for Injection(GlaxoSmithKline) and the melphalan formulation that containedSAE_(6.5)-β-CD. Specifically, the mean whole blood and plasmaconcentration versus time profile of melphalan with and withoutSBE_(6.5)-β-CD are essentially super-imposable. The results demonstratethat SBE_(6.5)-β-CD had no observable difference in blood or plasmaversus time profiles for melphalan in the rat model. Furthermore, therewas no apparent difference in the urinary excretion of melphalan in therat model.

Example 17

A Phase IIa, multicenter, open-label, randomized, efficacy and safetystudy of melphalan hydrochloride administered by injection using apropylene glycol-free diluent vehicle has been conducted in 3 humanmultiple myeloma patients who underwent myeloablative conditioning inpreparation for autologous transplantation. The study is on-going.

The primary goal of the study was/is to determine the rate ofmyeloablation and neutrophil engraftment in multiple myeloma patientswho receive a high-dose of melphalan hydrochloride via injection inwhich one dose is administered using a propylene glycol diluent and onedose is administered using a propylene glycol-free diluent. Theadministering is used as myeloablative therapy prior to autologous stemcell transplantation.

The secondary goal of the study is to determine: (a) the rate ofplatelet engraftment in multiple myeloma patients receiving high-dosemelphalan hydrochloride via injection using both a propylene glycoldiluent and a propylene glycol-free diluent prior to autologous stemcell transplantation; (b) the median time to engraftment of neutrophiland platelets in multiple myeloma patients receiving high-dose melphalanhydrochloride via injection using both a propylene glycol diluent and apropylene glycol-free diluent prior to autologous stem celltransplantation; (c) the response rate (stringent complete response[sCR], complete response [CR], very good partial response [VGPR],partial response [PR], stable disease [SD], or progressive disease [PD])at autologous stem cell transplantation at day +100 in multiple myelomapatients receiving high-dose melphalan hydrochloride via injection usinga propylene glycol diluent and a propylene glycol-free diluent prior toautologous stem cell transplantation; (d) the toxicity profile ofhigh-dose melphalan hydrochloride via injection using both a propyleneglycol diluent and a propylene glycol-free diluent in multiple myelomapatients undergoing autologous stem cell transplantation; (e) the rateof treatment-related mortality during the first 100 days afterautologous stem cell transplantation in multiple myeloma patientsreceiving high-dose melphalan hydrochloride via injection using apropylene glycol diluent and a propylene glycol-free diluent; and (f)the pharmacokinetics of melphalan hydrochloride via injection using apropylene glycol diluent compared with the pharmacokinetics of melphalanhydrochloride via injection using a propylene glycol-free diluent (i.e.,a cyclodextrin derivative) in multiple myeloma patients undergoingautologous stem cell transplantation.

Patients were screened prior to enrollment in the study. Patients fromany of the following classes were qualified for inclusion in the study:

Patients with symptomatic multiple myeloma requiring treatment atdiagnosis or anytime thereafter;

Patients with multiple myeloma who qualify for autologous stem celltransplantation therapy who have received appropriate primary inductiontherapy for transplantation;

Patients who are 70 years of age or younger at time of transplantation(patients greater than 70 years of age may qualify on a case-by-casebasis if the patient meets criterion based on institution's standard ofpractice);

Patients with an adequate autologous graft, defined as anun-manipulated, cryopreserved, peripheral blood stem cell or bone marrowstem cell graft containing at least 2×10⁶ CD34+ cells/kg based uponpatient weight, along with a reserve of 2×10⁶ CD34+ cells/kg that isstored in a separate bag; and

Patients with adequate organ function as measured by:

-   -   Cardiac: Left ventricular ejection fraction at rest >40%;    -   Hepatic: Bilirubin <2×the upper limit of normal and ALT/AST        <3×ULN;    -   Renal: Creatinine clearance >40 mL/minute; and    -   Pulmonary: DLCO, FEV₁, FVC >50% of predicted value (corrected        for Hgb) or O₂ saturation >92% on room air.

All patients have received antiemetics, hydration, and infectionprophylaxis according to institutional guidelines. Patients followedinstitutional guidelines regarding hospitalization. Patients returnedfor daily laboratory tests (CBC with differential and platelets and abasic chemistry panel) until neutrophil engraftment, and then returnedfor weekly safety evaluations until autologous stem cell transplantationDay +30. The following safety, efficacy, and pharmacokinetic evaluationswere performed prior to the first dose of melphalan, and at thefollowing post-dose time points:

-   -   Twelve blood samples were taken at specific time points for a        pharmacokinetic evaluation. Blood samples were collected        immediately prior to and after receiving the melphalan dose;    -   Vital signs were recorded hourly during the first eight hours        after receiving each dose of melphalan, then repeated once daily        until hospital discharge, and then weekly until Day +30. Weight        were collected at hospital discharge and at Day +30;    -   A 12-lead electrocardiograph, (ECG) along with a 10 to 20 second        rhythm strip was collected twice weekly until hospital        discharge, then a 12-lead ECG (without a rhythm strip) was        collected weekly until Day +30;    -   A focused physical examination was performed daily until        hospital discharge, then a complete physical examination was        performed weekly until Day +30;    -   Toxicity grading and evaluation for AEs/SAEs was according to        NCI-CTC AE Version 3.0 during the entire Study Period;    -   Complete blood count with differential and platelet count was        performed daily until neutrophil and platelet engraftment, then        weekly until Day +30;    -   Eastern Cooperative Group performance status was examined at the        time of hospital discharge, then weekly until Day +30;    -   Basic serum chemistry panel (sodium, potassium, chloride,        glucose, creatinine, bicarbonate, and BUN) daily until        neutrophil engraftment;    -   Full serum chemistry panel (sodium, potassium, chloride,        magnesium, bicarbonate, glucose, total protein, albumin,        calcium, phosphate, uric acid, BUN, creatinine, CPK, total        bilirubin, alkaline phosphatase, LDH, SGOT, and SGPT) will be        monitored weekly until Day +30;    -   Urinalysis (specific gravity, pH, protein, glucose, ketones,        nitrite, RBCs, and WBCs) was monitored twice weekly until        hospital discharge, then weekly until Day +30; and    -   Concomitant medications was recorded during the entire study        period.

A melphalan dose of 200 g/m² was divided into two separate, consecutivedoses of 100 mg/m² administered on two separate days (Day −3 and Day −2)prior to the patients receiving an autologous stem cell transplantation.For the calculation of body surface area, actual body weight was usedfor patients who weighed less than or between 100% to 130% of theirideal body weight. Patients who weighed more than 130% of their idealbody weight were dosed based on a body surface area obtained bycalculating the patient's adjusted body weight.

Patients were randomly chosen to receive the first melphalan dose of 100mg/m² (on Day −3) via either a composition comprising a propylene glycoldiluent (i.e., Melphalan HCl Injectable, Bioniche Pharma USA) or acomposition comprising a cyclodextrin derivative (SBE_(6.5)-β-CD,CAPTISOL®, at a concentration of 125 mM). Patients who randomly receivedthe first melphalan dose of 100 mg/m² as a composition comprising acyclodextrin derivative (on Day −3) then received a second melphalandose of 100 mg/m² (on Day −2) using the composition comprising apropylene glycol diluent. Conversely, patients who randomly received thefirst melphalan dose of 100 mg/m² as a composition comprising apropylene glycol diluent (on Day −3) then received a second melphalandose of 100 mg/m² (on Day −2) using the composition comprising acyclodextrin derivative.

For the composition comprising a cyclodextrin derivative, a dry powdercomposition containing melphalan as a hydrochloride salt was dilutedwith normal saline to a melphalan concentration no greater than 0.45mg/mL and a cyclodextrin concentration of 125 mM. The diluted solutionwas infused over 60 minutes via a central venous catheter.

The composition comprising a propylene glycol diluent was administeredusing a cyclodextrin-free composition (Melphalan HCl Injectable,Bioniche Pharma USA) using the protocol described herein supra.

Following one day of rest after the myeloablative conditioning (Day −1),patients received an autologous graft with a minimum cell dose of 2×10⁶CD34+ cells/kg of patient body weight (Day 0). Cryopreservation andthawing of the product was consistent with Foundation for theAccreditation of Cellular Therapy standards and local institutionalpractice. The graft was infused per institutional protocol. Starting onDay +5, G-CSF was be administered at a dose of 5 μg/kg/day untilabsolute neutrophil count was greater than 500/mm³.

Blood samples for pharmacokinetic evaluation of melphalan were collectedafter each dose of melphalan and the pharmacokinetic parameters for invivo melphalan distribution were evaluated. Samples for evaluation ofthe pharmacokinetic parameters were collected by taking 5 mL venousblood samples immediately prior to melphalan administration and at 0,10, 20, 30, 60, 90, 120, 180, 240, 360, and 480 minutes following theend of the melphalan infusion. Pharmacokinetic parameters weredetermined by nonparametric pharmacokinetic data analysis techniques.Pharmacokinetic parameters computed from plasma drug concentration-timedata include the following:

-   -   C_(max), derived from the individual raw data;    -   T_(max), derived from the individual raw data;    -   Apparent terminal first-order elimination rate constant        (k_(e1));    -   Apparent elimination t_(1/2);    -   Area under the plasma concentration-time curve to the last        measurable time point (AUC_(0-t)), calculated by the trapezoidal        rule; and    -   Area under the plasma concentration-time curve from the last        measurable time point extrapolated to infinity (AUC_(t-∞)),        determined from the concentration at the last measurable time        point divided by the k_(e1).

The plasma concentrations and pharmacokinetic parameters were summarizedusing descriptive statistics. The data from patients 1-3 is shown in thefollowing table.

TABLE Individual patient melphalan pharmacokinetic parameters afterintravenous administration of a melphalan formulation that containedSBE_(6.5)-β-CD and a cyclodextrin-free melphalan formulation (i.e.,Melphalan HCl Injectable, Bioniche Pharma USA). SBE_(6.5)-β-CD CD-freePatient Parameter (27% w/v) formulation Ratio 1^(a) C_(max) (ng/mL)3,230 2,160 1.50 T_(max) (min) 10 20 — AUC_(0-t) 259,073 202,714 1.28AUC_(0-∞) 264,656 208,028 1.27 λ_(z) (min⁻¹) 0.0105 0.0103 — t_(1/2)(min) 65.8 67.1 — 2^(b) C_(max) (ng/mL) 2,730 2,010 1.36 T_(max) (min)10 10 — AUC_(0-t) 198,051 151,456 1.31 AUC_(0-∞) 202,728 154,130 1.32λ_(z) (min⁻¹) 0.0103 0.0113 — t_(1/2) (min) 67.4 61.6 — 3^(a) C_(max)(ng/mL) 4,590 2,890 1.59 T_(max) (min) 10 10 — AUC_(0-t) 306,432 230,6811.33 AUC_(0-∞) 314,108 236,059 1.33 λ_(z) (min⁻¹) 0.0101 0.0104 —t_(1/2) (min) 68.5 66.4 — ^(a)Patients 1 and 3 were administered theSBE_(6.5)-β-CD-containing formulation on Day −3 and the CD-freeformulation on Day −2. ^(b)Patient 2 was administered the CD-freeformulation on Day −3 and the SBE_(6.5)-β-CD-containing formulation onDay −2.

FIG. 6, provides a graphic representation of the mean plasma melphalanconcentration in a human patient after intravenous administration of amelphalan formulation containing a cyclodextrin derivative(SBE_(6.5)-β-CD) and after intravenous administration of acyclodextrin-free melphalan formulation (Melphalan HCl Injectable,Bioniche Pharma USA). Referring to FIG. 6 and the data in the abovetable, the in vivo distribution of melphalan administered with asulfoalkyl ether cyclodextrin derivative provides a nearly 50% increasein the maximum in vivo concentration of melphalan, and approximately a30% increase in the area under the plasma concentration curves (i.e.,for both AUC_(0-t) and AUC_(0-∞)). As shown in the table above, the datafor patients 2 and 3 exhibited similar pharmacokinetic results. In viewof pharmacokinetic data obtained for these melphalan formulations in therat model, the enhancement in C_(max) and AUC for theSBE_(6.5)-β-CD-containing melphalan formulation in human patients iswholly unexpected.

As noted above, the study is on-going. The primary efficacy end points,which will be based on an intent-to-treat analysis of all patients, willbe the rate of myeloablation and the rate neutrophil engraftment. Thefollowing definitions will be used for these end points:

Myeloablation will be defined as any of the following:

-   -   Absolute neutrophil count less than 0.5×10⁹/L;    -   Absolute lymphocyte count less than 0.1×10⁹/L; or    -   Platelet count less than 20,000/mm³ or bleeding requiring        transfusion.

The first of two consecutive days for which cell counts drop below thesecut-off levels will be recorded as the date of myeloablation.

Neutrophil engraftment is defined as absolute neutrophil count greaterthan 0.5×10⁹/L on three consecutive daily assessments.

Secondary efficacy end points will be based on the following criteria:

-   -   The rate of platelet engraftment, which will be defined as an        un-transfused platelet measurement >20,000/mm³ on three        consecutive daily assessments;    -   The time to neutrophil engraftment, which will be defined as the        first of three assessments where absolute neutrophil count is        greater than 0.5×10⁹/L;    -   The time to platelet engraftment, which will be defined as the        first of three consecutive daily assessments where un-transfused        platelet measurement is greater than 20,000/mm³;    -   The rate of non-engraftment, which will be defined as a failure        to reach an absolute neutrophil count greater than 0.5×10⁹/L on        three consecutive daily assessments by autologous stem cell        transplantation Day +100;    -   The rate of late graft failure or late rejection, which will be        defined as development of absolute neutrophil count less than        0.5×10⁹/L after having engrafted within the first 100 days;    -   The rate of multiple myeloma response (sCR, CR, VGPR, PR, SD, or        PD), which will be defined according to International Working        Group criteria at Day +100; and    -   The rate of treatment-related mortality, which will be defined        as death without relapse or progression at Day +100.

The clinical trial is expected to demonstrate that melphalanadministered with a cyclodextrin derivative (SBE_(6.5)-β-CD) istherapeutically effective and safe for use in subjects for whom a stemcell transplantation has been indicated as conditioning prior to stemcell transplantation.

Example 18

A Phase IIb, multi-center, open-label, non-randomized, efficacy andsafety study of melphalan hydrochloride administered by injection usinga propylene glycol-free vehicle will be conducted in human multiplemyeloma patients who have symptomatic multiple myeloma and qualify forASCT.

The parameters of the study will be similar to those described inExample 17, except that all patients will be administered a propyleneglycol-free melphalan composition (100 mg/m²) on Day −3 and Day −2 usinga melphalan composition that includes a cyclodextrin derivative.Otherwise, the inclusion criteria, exclusion criteria, safety criteria,dosing, treatment, and efficacy endpoints will be similar to thosedescribed above in Example 17.

CONCLUSION

These examples illustrate possible embodiments of the present invention.While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. It will be apparent to persons skilledin the relevant art that various changes in form and detail can be madetherein without departing from the spirit and scope of the invention.Thus, the breadth and scope of the present invention should not belimited by any of the above-described exemplary embodiments, but shouldbe defined only in accordance with the following claims and theirequivalents.

All documents cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedor foreign patents, or any other documents, are each entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited documents.

1. A method of treating a subject suffering from a neoplastic disorder,the method comprising: diluting a composition with an aqueous diluent toprovide a dilute pharmaceutical composition comprising 25 mg to 125 mgof melphalan and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thedilute pharmaceutical composition has a pH of about 4 to about 6;wherein the cyclodextrin derivative is present in a concentration of atleast 50:1 (w/w) relative to the melphalan; wherein the melphalan in thedilute pharmaceutical composition degrades by 2% or less at about 25° C.within 5 hours after the diluting; and administering the dilutepharmaceutical composition by injection to the subject in need thereof.2. The method of claim 1, wherein the neoplastic disorder is selectedfrom: myeloma, multiple myeloma, melanoma, acute myelogenous leukemia,malignant melanoma, breast cancer, ovarian cancer, testicular cancer,advanced prostate cancer, a neuroendocrine cancer, metastatic melanoma,a metastatic neuroendocrine tumor, a metastatic adenocarcinoma tumor,hepatocellular carcinoma, osteogenic sarcoma, polycythemia veraplasma,plasma cell neoplasm, amyloidosis, scleromyxedema, and combinationsthereof.
 3. The method of claim 2, wherein the neoplastic disorder ismultiple myeloma and the administering is systemic and providespalliative treatment of the multiple myeloma.
 4. The method of claim 1,wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is ahydroxy-substituted-C₃ group.
 5. The method of claim 1, wherein R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are independently a straight-chain orbranched C₁-C₈-(alkylene)-SO₃ ⁻ group having a degree of substitution of4 to 8 per cyclodextrin derivative, and the remaining substituents are—H.
 6. The method of claim 1, wherein at least one of R₁, R₂, R₃, R₄,R₅, R₆, R₇, R₈ and R₉ is substituted with a straight-chainC₄-(alkylene)-SO₃ ⁻ group.
 7. The method of claim 1, wherein thecyclodextrin derivative is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; whereinthe pharmaceutical composition comprises about 50 mg of melphalan as ahydrochloride salt; and wherein the cyclodextrin derivative is presentin a concentration of 50:1 to 100:1 (w/w) relative to the melphalan. 8.The method of claim 1, wherein the dilute pharmaceutical composition issubstantially free of an alcohol.
 9. The method of claim 1, wherein theaqueous diluent is a saline solution.
 10. The method of claim 1, whereinthe subject suffering from the neoplastic disorder is a pediatricsubject.
 11. The method of claim 1, wherein the melphalan in the dilutepharmaceutical composition degrades by 4% or less at 25° C. within 10hours after the diluting.
 12. The method of claim 1, wherein the dilutepharmaceutical composition is stored about 0.5 hours to about 18 hoursprior to the administering.
 13. The method of claim 1, wherein theadministering provides a melphalan C_(max) in the subject suffering froma neoplastic disorder that is at least 20% or greater than a melphalanC_(max) provided by a melphalan formulation containing an equivalentdose of melphalan and lacking the cyclodextrin derivative.
 14. Themethod of claim 1, wherein the administering provides a melphalanAUC_(0-t) in the subject suffering from a neoplastic disorder that is atleast 20% or greater than a melphalan AUC_(0-t) provided by a melphalanformulation containing an equivalent dose of melphalan and lacking thecyclodextrin derivative.
 15. A method for conditioning a subject forwhom a stem cell transplantation has been indicated, the methodcomprising: administering a melphalan dose of 50 mg/m² to 300 mg/m² perday to the subject for whom a stem cell transplantation has beenindicated, wherein the melphalan dose is administered in apharmaceutical composition comprising melphalan and a cyclodextrinderivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6; and whereinthe cyclodextrin derivative is present in a ratio of at least 25:1 (w/w)relative to the melphalan.
 16. The method of claim 15, wherein theadministering is for a period of two or more days.
 17. The method ofclaim 15, wherein the subject in need of the stem cell transplantationis a pediatric subject.
 18. The method of claim 15, wherein theadministering is performed intravenously.
 19. The method of claim 15,wherein the administering is performed via a limb perfusion.
 20. Themethod of claim 15, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇,R₈ and R₉ is a hydroxy-substituted-C₃ group.
 21. The method of claim 15,wherein the cyclodextrin derivative is a compound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; whereinthe pharmaceutical composition comprises about 200 mg of melphalan as ahydrochloride salt; and wherein the cyclodextrin derivative is presentin a ratio of 25:1 to 35:1 (w/w) relative to the melphalan.
 22. Themethod of claim 15, wherein the subject for whom a stem celltransplantation has been indicated suffers from a disease or disorderselected from: myeloma, multiple myeloma, a lymphoma, non-Hodgkinlymphoma, leukemia, acute myeloid leukemia, Hodgkin's disease, acutelymphoblastic leukemia, a myelodysplastic syndrome, a myeloproliferativedisorder, chronic myelogenous leukemia, neuroblastoma, aplastic anemia,chronic granulocytic leukemia, a neuroblastoma, sickle-cell disease,osteogenic sarcoma, Ewing's sarcoma, a desmoplastic small round celltumor, plasma cell neoplasm, amyloidosis, scleromyxedema, andcombinations thereof.
 23. The method of claim 15, comprising diluting aconcentrated melphalan composition with an aqueous diluent to providethe pharmaceutical composition.
 24. The method of claim 23, wherein theconcentrated melphalan composition comprises 50 mg to 500 mg ofmelphalan.
 25. The method of claim 23, wherein the concentratedmelphalan composition comprises about 200 mg.
 26. The method of claim15, wherein the pharmaceutical composition is substantially free of analcohol.
 27. The method of claim 23, wherein the aqueous diluent is asaline solution.
 28. The method of claim 23, wherein the melphalan inthe pharmaceutical composition degrades by 4% or less at about 25° C.within 10 hours after the diluting.
 29. The method of claim 23, whereinthe pharmaceutical composition is stored about 0.5 hours to about 12hours prior to the administering.
 30. The method of claim 15, whereinthe administering provides a melphalan C_(max) in the subject for whom astem cell transplantation has been indicated that is at least 20% orgreater than a melphalan C_(max) provided by a melphalan formulationcontaining an equivalent dose of melphalan and lacking the cyclodextrinderivative.
 31. The method of claim 15, wherein the administeringprovides a melphalan AUC_(0-t) in the subject for whom a stem celltransplantation has been indicated that is at least 20% or greater thana melphalan AUC_(0-t) provided by a melphalan formulation containing anequivalent dose of melphalan and lacking the cyclodextrin derivative.32. A pharmaceutical composition comprising: 25 mg to 125 mg ofmelphalan as a hydrochloride salt; an optional buffer; and acyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6, whereindilution of the pharmaceutical composition with an aqueous solutionprovides a dilute pharmaceutical composition in which the melphalandegrades by 2% or less at about 25° C. within 5 hours after thedilution; and wherein the cyclodextrin derivative is present in a ratioof 50:1 to 100:1 (w/w) relative to the melphalan.
 33. The pharmaceuticalcomposition of claim 32, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆,R₇, R₈ and R₉ is a hydroxy-substituted-C₃ group.
 34. The pharmaceuticalcomposition of claim 32, wherein the cyclodextrin derivative is acompound of formula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; whereinthe pharmaceutical composition comprises about 50 mg of melphalan as ahydrochloride salt; and wherein the cyclodextrin derivative is presentin a ratio of about 55:1 (w/w) relative to the melphalan.
 35. Apharmaceutical composition comprising: 150 mg to 250 mg of melphalan asa hydrochloride salt; an optional buffer; and a cyclodextrin derivativeof formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thepharmaceutical composition has a pH of about 4 to about 6, whereindilution of the pharmaceutical composition with an aqueous solutionprovides a melphalan solution in which the melphalan degrades by 2% orless at about 25° C. within 5 hours after the dilution; and wherein thecyclodextrin derivative is present in a ratio of 25:1 to 35:1 (w/w)relative to the melphalan.
 36. The pharmaceutical composition of claim35, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is ahydroxy-substituted-C₃ group.
 37. The pharmaceutical composition ofclaim 35, wherein the cyclodextrin derivative is a compound of formulaII:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; whereinthe pharmaceutical composition comprises about 200 mg of melphalan as ahydrochloride salt; and wherein the cyclodextrin derivative is presentin a ratio of about 30:1 (w/w) relative to the melphalan.
 38. Apharmaceutical kit comprising: a first container comprising 25 mg to 125mg of melphalan as a hydrochloride salt and an optional water-solublepolymer; and a second container comprising an aqueous diluent, anoptional buffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of at least 50:1 (w/w) relative to the melphalan; andwherein combining the first container and the second container providesa dilute pharmaceutical composition having a pH of about 4 to about 6that degrades by 2% or less at about 25° C. within 5 hours after thediluting.
 39. The pharmaceutical kit of claim 38, wherein the firstcontainer comprises povidone in an amount of 10 mg to 30 mg, and thesecond container comprises a pH-adjusting agent in a concentrationsufficient to provide a pH of about 4 to about 6 when the firstcontainer and the second container are combined.
 40. A pharmaceuticalkit comprising: a first container comprising 150 mg to 250 mg ofmelphalan as a hydrochloride salt and an optional water-soluble polymer;and a second container comprising an aqueous diluent, an optionalbuffer, and a cyclodextrin derivative of formula I:

wherein n is 4, 5 or 6; wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉are independently —H, a straight-chain or branched C₁-C₈-(alkylene)-SO₃⁻ group, or an optionally substituted straight-chain or branched C₁-C₆group; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isa straight-chain or branched C₁-C₈-(alkylene)-SO₃ ⁻ group; wherein thecyclodextrin derivative is present in the second container in aconcentration of 25:1 to 35:1 (w/w) relative to the melphalan; andwherein combining the first container and the second container providesa dilute pharmaceutical composition having a pH of about 4 to about 6that degrades by 2% or less at about 25° C. within 5 hours after thediluting.
 41. The pharmaceutical kit of claim 40, wherein the firstcontainer comprises povidone in an amount of 10 mg to 30 mg, and thesecond container comprises a pH-adjusting agent in a concentrationsufficient to provide a pH of about 4 to about 6 when the firstcontainer and the second container are combined.
 42. The pharmaceuticalkits of claims 40, wherein the cyclodextrin derivative is a compound offormula II:

wherein R═(H)_(21-x) or (—(CH₂)₄—SO₃ ⁻Na⁺)_(x), and x=6.0-7.1; whereinthe first container comprises about 200 mg of melphalan as ahydrochloride salt; and wherein the cyclodextrin derivative is presentin the second container in an amount of about 30:1 (w/w) relative to themelphalan.